Novel compounds and process

ABSTRACT

The present invention relates to a novel chemical process for the covalent conjugation of disulphide bridge cyclised peptides to immunogenic carrier molecules by thio-ether linkages to form vaccine immunogens. In particular, the novel chemistry involves reacting a thiolated carrier with a cyclic peptide containing a disulphide bridge, which cylcic peptide (herein a disulphide bridge cyclised peptide) has attached to it, usually via a linker, a reactive group capable for forming thio-ether bonds with the carrier. The invention further related to activated peptide intermediates of the process, medicaments produced by the process, pharmaceutical compositions containing the medicaments, and the use of the pharmaceutical compositions in medicine. The process of the present invention is particularly useful for the preparation of highly pure immunogens for vaccines, comprising disulphide bridge cyclised peptides. Also novel immunogens are provided, base don peptides derived from the sequence of human IgE, which are useful in the immunotherapy of allergy. Accordingly, the inventions related also to a process for conjugation of IgE disulphide bridge cyclised peptides to carrier, immunogens produced by the process and vaccines and pharmaceutical compositions comprising them and their use in the treatment of allergy.

[0001] The present invention relates to a novel chemical process for thecovalent conjugation of disulphide bridge cyclised peptides toimmunogenic carrier molecules by thio-ether linkages to form vaccineimmunogens. In particular, the novel chemistry involves reacting athiolated carrier with a cyclic peptide containing a disulphide bridge,which cyclic peptide (herein a disulphide bridge cyclised peptide) hasattached to it, usually via a linker, a reactive group capable offorming thio-ether bonds with the carrier. The invention further relatesto activated peptide intermediates of the process, medicaments producedby the process, pharmaceutical compositions containing the medicaments,and the use of the pharmaceutical compositions in medicine. The processof the present invention is particularly useful for the preparation ofhighly pure immunogens for vaccines, comprising disulphide bridgecyclised peptides. Also novel immnunogens are provided, based onpeptides derived from the sequence of human IgE, which are useful in theimmunotherapy of allergy. Accordingly, the invention relates also to aprocess for conjugation of IgE disulphide bridge cyclised peptides tocarriers, immunogens produced by the process and vaccines andpharmaceutical compositions comprising them and their use in thetreatment of allergy.

[0002] Immunogens comprising short peptides are becoming increasinglycommon in the field of vaccine prophylaxis or therapy. In many diseasestates it is often possible, and desirable, to design vaccinescomprising a short peptide rather than a large protein. Peptides whichmay be used as immunogens may be the full length native protein, forexample human peptidic hormones, or may be fragments of a larger antigenderived from a given pathogen, or from a large self-protein. Forexample, short peptides of IgE may be used for prophylaxis of allergy,whereas the use of IgE itself as the immunogen may induce anaphylacticshock.

[0003] It has previously been thought that amongst the problemsassociated with the peptide approach to vaccination, is the fact thatpeptides per se are poor immunogens. Generally the sequences of thepeptides chosen are such that they include a B-cell epitope to provide atarget for the generation of anti-peptide antibody responses, butbecause of their limited size rarely encompass sufficient T-cellepitopes in order to provide the necessary cytokine help in theinduction of strong immune responses following priming and boostingapplications of the vaccine.

[0004] Strategies to overcome this problem of immunogenicity include thelinking of the peptide to large highly immunogenic protein carriers. Thecarrier proteins contain a large number of peptidic T-cell epitopeswhich are capable of being loaded into MHC molecules, thereby providingbystander T-cell help, and/or alternatively the use of strong adjuvantsin the vaccine formulation. Examples of these highly immunogeniccarriers which are currently commonly used for the production of peptideimmunogens include the Diptheria and Tetanus toxoids (DT and TTrespectively), Keyhole Limpet Haemocyanin (KLH), and the purifiedprotein derivative of Tuberculin (PPD).

[0005] Peptides used in a particular vaccine immunogen are often chosensuch that they generate an antibody response to the location site ofthat peptide in the context of the full length native protein. Thus, inorder to generate antibodies that bind to such chosen locations, thepeptide in the immunogen must assume substantially the same shape as itwould exist if it was confined by the flanking regions of the fulllength native protein. However, merely conjugating a linear peptidesequence, by conventional chemistry, to a carrier protein rarelyachieves this goal. This is because such an immunogen presents thelinear peptide with too much conformational freedom, such that thepeptide may adopt a loose structure that either is not well recognisedby the immune system, or may be entirely different to the conformationadopted by the peptide in the context of the flanking regions of thefull length native protein.

[0006] In order to overcome this conformational freedom problem, it isknown to design peptides in a constrained manner, by chemicalinteractions between two distant amino acid residues, such that thepeptide is held in a curved structure which closely resembles the curvein which the peptide would be held by the flanking sequences in the fulllength native protein (U.S. Pat. No. 5,939,383; Hruby et al., 1990,Biochem J., 268, 249-262). To do this it is most common to incorporatetwo cysteine residues into the peptide sequence between which thedesired intramolecular disulphide bridge forms after gentle oxidation ofa dilute solution of the peptide.

[0007] The cyclised peptide thus formed is commonly conjugated to aprotein carrier to form an immunogen by one of several chemistrymethods. Examples of known chemistries include conjugation of aminogroups between the peptide and carrier by amino reactive agents such asglutaraldehyde or formaldehyde; or condensing carboxyl groups and aminogroups with carbodiimide reagents or alternatively by convertingn-terminal (x-hydroxy groups to aldehydes by an oxidation reaction andconjugating this group to an amino or oxamino moiety. However, each ofthese chemistries has disadvantages, including a need for relativelyharsh oxidative reaction conditions, poor controllability at industriallevels, formation of polymers, or not being suitable for peptides thatcontain specific internal amino acids (especially: Lysine, Asparticacid, Glutamic acid, Tryptophan, Tyrosine or Serine) that could alsointerfere with the chemistry in an inappropriate manner.

[0008] It is common, therefore, to use thio-ether linkage to conjugatepeptides to protein carriers. The most common method to achieve thisconjugation is to add a moiety with a terminal thiol group onto thepeptide, most commonly by adding a cysteine, and then to react thereactive thiol group with a maleimide-derivatised protein carrier(Friede et al., 1994, Vaccine, 12, 791-797), for a schematic summary seeFIG. 1.

[0009] However, in the case of peptides containing an internaldisulphide bond this commonly preferred peptide chemistry may haveproblems because of the posibility of internal disulphide rearrangement,or external rearrangement of disulphide bonds between between twoadjacent peptides. In some cases the presence of a third cysteine causesunwanted interference with the disulphide bond, and a thiol-disulfideexchange can occur such that the resultant intermediate cyclised peptideproduct is a mixture of three possible disulphide bridge cyclisedpeptides (reassortant intermediates, see FIG. 2), or may additionallycomprise peptide dimers or polymers.

[0010] In the case of conjugation of these peptide intermediates to amaleimide activated carrier protein, each of the reassortantintermediates is equally reactive with the reactive carrier protein, andas such they will all conjugate to the carrier. As a result, the purityof the desired product is decreased, and use of this mixture ofimmunogens may result in immune responses that may not, or only weakly,cross react with the epitope on the full native protein that the peptidewas intended to mimic. In order to overcome these problems severalauthors have replaced the disulphide bond stabilised cyclic peptides, bythio-ether bonds. For example, in Ivanov et al., 1995, BioconjugateChemistry, 6, 269-277, one cystein is replaced by a trifunctionalbromoacetyl-derivitised amino acid, thus permiting cyclisation via anon-reversible thioether bond. In such thio-ether cyclised peptides,however, the resulting peptide is fundamentally different to theoriginal disulphide-cyclised peptide, and has a different structurewhich may not resemble the disulphide-cyclised peptide. Hence antibodiesformed against the thio-ether cyclised peptide may not recognise theparent peptide as efficiently as antibodies formed against thedisulphide-cyclised peptides.

[0011] The present invention overcomes the problems of forming athio-ether linkage between a disulphide cyclised peptide and a carrierby providing a chemistry that does not use a terminal thiol containinggroup on the cyclised peptide, but instead uses another reactive groupon the peptide, which may then be reacted with a thiolated carrierprotein to form a thio-ether bond.

[0012] Therefore, in the present invention, there is provided a processfor the manufacture of a vaccine immunogen comprising conjugating adisulphide bridge cyclised peptide to an immunogenic carrier comprising,(a) adding to a disulphide cyclised peptide a moiety comprising areactive group which is capable of forming thio-ether linkages withthiol bearing carriers, and (b) reacting the activated cyclised peptidethus formed with a thiol bearing immunogenic carrier.

[0013] The process of the present invention overcomes the problems ofinternal and external disulphide rearrangement, and in addition providesconjugated products wherein the disulphide cyclised peptides are in thedesired conformation. In a preferred process of the present invention, apeptide is synthesised containing two cysteine residues which areallowed to form a disulphide bridge, followed by the addition of thereactive group. The activated peptide, thus obtained, is then reactedwith the thiol bearing carrier.

[0014] The reactive groups that are suitable for use in the presentinvention include any group which is capable forming thio-ether linkageswith thiolated carriers. As which will be apparent to the man skilled inthe art, preferred reactive groups may be selected from active imides,especially maleimides, haloalkyl groups such as iodoalkyl or bromoalkylgroups. Preferably the bromoalkyl group is a bromoacetyl group. The useof maleimide to link linear peptides to thiolated polymer is describedin Van Dijk-Wolthius et al., 1999, Bioconjugate Chemistry, 10, 687-692.Use of bromoacetyl groups to link peptides to carriers is described inIvanov et al., 1995, Bioconjugate Chemistry, 6, 269-277 and U.S. Pat.No. 5,444,150. Conjugation of proteins to thiolated solid phase supportsfor diagnostic assays is described in EP 0 396 116 A.

[0015] It is a particularly preferred aspect of the present inventionwhen the process uses maleimide as the reactive group. Accordingly, apreferred process for conjugating a disulphide bridge cyclised peptideto a carrier comprises, (a) adding to a disulphide cyclised peptide amoiety comprising a maleimide group, and (b) reacting the activatedcyclised peptide thus formed with a thiol bearing carrier. The productof this process (A conjugate suitable for use in a vaccine) forms anaspect of the present invention, and has the formula (I):

[0016] wherein, Carrier is a carrier molecule, X is either a linker or abond, Y is either a linker or a bond, and P is a disulphide bridgecyclised peptide. When X is a bond, it should be understood that thecarrier is directly linked to the sulphur atom S. Similarly, when Y is alinker it should be understood that the disulphide-bridge cyclisedpeptide is linked directly to the nitrogen atom N. A “linker” refers toa suitable linker group. When X is a linker group an example is thegroup —NHCO(CH₂)₂—. When Y is a linker group, an example is—(CH₂)₃—CONH—. It will also be clear to the man skilled in the art, thatFormula (I) covers conjugates where the sulphur atom (S) is joined ontothe imide ring to either of the two adjacent non-carbonyl carbon atoms,such that the conjugate may comprise the following structures:

[0017] Forming an aspect of the invention is the intermediate to theprocess of the present invention, which is a disulphide cyclised peptidewhich bears a reactive group which is capable forming thio-etherlinkages with thiolated carriers. Preferably said intermediate comprisesa disulphide bridge cyclised peptide linked to an active imide group, inparticular a maleimide group. The high purity of the final conjugatedproduct derives from the fact that any internal or externalrearrangement that occurs between the disulphide bridge and thethio-ether reactive group is irreversible, and consequently thesereassortant intermediates are not reactive with the thiolated carrierprotein. Only the activated peptide intermediates that have thedisulphide bridge at the desired location (i.e. between the cysteinespresent in the peptide) with the free reactive group participate in theconjugation reaction with the thiolated carrier, thereby forming aconjugate of extremely high purity which contains cyclised peptides ofthe desired conformation.

[0018] Preferred maleimide derivatisation reagents aregamma-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS, MolecularFormula: C₁₂H₁₂N₂O₆ Fujiwara, K., et al., J. Immunol. Meth., 45, 195-203(1981), Tanimori, H., et al., J. Pharmacobiodyn., 4, 812-819 (1981); H.Tanimori, et al., J. Immunol. Methods 62, 123 (1983); M.D. Partis, etal., J. Prot. Chem. 2, 263 (1983); L. Moroder, et al., Biopolymers 22,481 (1983); S. Hashida, et al., J. Appl. Biochem. 6, 56 (1984); S.Inoue, et al., Anal. Lett. 17, 229 (1984); E. Wünsch, et al., Biol.Chem. Hoppe-Seyler 366, 53 (1985)), which can be purchased from theSigma or Pierce companies. It will be recognised that manymaleimide-derivitisation reagents exist and can be used, and theaddition of the maleimide group to the cyclised peptide can be performedduring peptide synthesis using reagents compatible with organicsynthesis, or after peptide synthesis using reagents commonly used forderivitising peptides and proteins with maleimide groups.

[0019] The process, intermediates and products of the present inventionare preferably used in the manufacture of immnunogens for use invaccines. The peptides for conjugation may be selected from any antigenagainst which is desired to create an immune response. The peptide maybe derived from a pathogen, such as a virus, bacterium, parasite such asa worm etc.

[0020] Equally the peptide may be selected from a self protein, forexample in the vaccine therapy of cancer or allergy.

[0021] In an allergic response, the symptoms commonly associated withallergy are brought about by the release of allergic mediators, such ashistamine, from immune cells into the surrounding tissues and vascularstructures. Histamine is normally stored in mast cells and basophils,until such time as the release is triggered by interaction with allergenspecific IgE. The role of IgE in the mediation of allergic responses,such as asthma, food allergies, atopic dermatitis, type-Ihypersensitivity and allergic rhinitis, is well known. On encounteringan antigen, such as pollen or dust mite allergens, B-cells commence thesynthesis of allergen specific IgE. The allergen specific IgE then bindsto the FcεRI receptor (the high affinity IgE receptor) on basophils andmast cells. Any subsequent encounter with allergen leads to thetriggering of histamine release from the mast cells or basophils, bycross-linking of neighbouring IgE/FcεRI complexes (Sutton and Gould,Nature, 1993, 366: 421-428; EP 0 477 231 B1).

[0022] IgE, like all immunoglobulins, comprises two heavy and two lightchains. The ε heavy chain consists of five domains: one variable domain(VH) and four constant domains (Cε1 to Cε4). The molecular weight of IgEis about 190,000 Da, the heavy chain being approximately 550 amino acidsin length. The structure of IgE is discussed in Padlan and Davis (Mol.Immunol., 23, 1063-75, 1986) and Helm et al., (2IgE model structuredeposited Feb. 2, 1990 with PDB (Protein Data Bank, ResearchCollabarotory for Structural Bioinformatics;http:pdb-browseres.evi.ac.uk)). Each of the IgE domains consists of asquashed barrel of seven anti-parallel strands of extended (β-)polypeptide segments, labelled a to f, grouped into two β-sheets. Fourβ-strands (a, b,d & e) form one sheet that is stacked against the secondsheet of three strands (c,f & g) (see FIG. 8). The shape of each β-sheetis maintained by lateral packing of amino acid residue side-chains fromneighbouring anti-parallel strands within each sheet (and is furtherstabilised by main-chain hydrogen-bonding between these strands). Loopsof residues, forming non-extended (non-β-) conformations, connect theanti-parallel β-strands, either within a sheet or between the opposingsheets. The connection from strand a to strand b is labelled as the A-Bloop, and so on. The A-B and d-e loops belong topologically to thefour-stranded sheet, and loop f-g to the three-stranded sheet. Theinterface between the pair of opposing sheets provides the hydrophobicinterior of the globular domain. This water-inaccessible, mainlyhydrophobic core results from the close packing of residue side-chainsthat face each other from opposing β-sheets.

[0023] In the past, a number of passive or active immunotherapeuticapproaches designed to interfere with IgE-mediated histamine releasemechanism have been investigated. These approaches include interferingwith IgE or allergen/IgE complexes binding to the FcεRI or FcεRII (thelow affinty IgE receptor) receptors, with either passively administeredantibodies, or with passive administration of IgE derived peptides tocompetitively bind to the receptors. In addition, some authors havedescribed the use of specific peptides derived from IgE in activeimmunisation to stimulate histamine release inhibiting immune responses.

[0024] Therefore, in order to be effective, the peptide vaccines need tobe able to mimic specific sites of IgE very efficiently. The preferredimmunogens of the present invention, therefore, are based on peptidesderived from IgE and which are capable of triggering an immune responsewhich inhibits histamine release from basophils.

[0025] Much work has been carried out to identify specific anti-IgEantibodies which do have some beneficial effects against IgE-mediatedallergic reaction (WO 90/15878, WO 89/04834, WO 93/05810). Attempts havealso been made to identify epitopes recognised by these usefulantibodies, to create peptide mimotopes of such epitopes and to usethose as immunogens to produce anti-IgE antibodies.

[0026] WO 97/31948 describes an example of this type of work, andfurther describes IgE peptides from the Cε3 and Cε4 domains conjugatedto carrier molecules for active vaccination purposes. These immunogensmay be used in vaccination studies and are said to be capable ofgenerating antibodies which subsequently inhibit histamine release invivo. In this work, a monoclonal antibody (BSW17) was described whichwas said to be capable of binding to IgE peptides contained within theCε3 domain which are useful for active vaccination purposes.

[0027] EP 0 477231 B 1 describes immunogens derived from the Cε4 domainof IgE (residues 497-506, also known as the Stanworth decapeptide),conjugated to Keyhole Limpet Haemocyanin (KLH) used in activevaccination immunoprophylaxis. WO 96/14333 is a continuation of the workdescribed in EP 0 477 231 B 1.

[0028] Other approaches are based on the identification of peptidesderived from Cε3 or Cε4, which themselves compete for IgE binding to thehigh or low affinity receptors on basophils or mast cells (WO 93/04173,WO 98/24808, EP 0 303 625 B1, EP 0 341290).

[0029] Accordingly in a preferred aspect of the present invention theprocess, peptide intermediates, immunogens and vaccines, comprise apeptide selected from human IgE. Preferably the disulphide bridgecyclised peptides used in the present invention are designed from thegroup of peptides listed in table 1. The peptides in table 1, reflect aspecific area of the IgE molecule against which it is desired togenerate an immune response. The peptides, therefore, constitute astarting point from which a cyclised peptide may be designed, andaccordingly they either do not contain a cysteine residue, or contain asingle cysteine, or contain two cysteines which may not form adisulphide bridge. Suitable peptides for use in the process orimmunogens of the present invention may be designed by the addition ofat least one cysteine residue to the following peptides: TABLE 1 IgEpeptides suitable to be cyclised and used in the process of the presentinvention Peptide sequence SEQ ID NO. EDGQVMDVD 1 STTQEGEL 2 SQKHWLSDRT3 GHTFEDSTKK 4 GGGHFPPT 5 PGTINI 6 FTPPT 7 CLEDGQVMDVDLL 8 LLDVDMVQGDELC9 WLEDGQVMDVDLC 10 QVMDVDL 11 LEDGQVMDVD 12 CSTTQEGELA 13 TTQEGE 14CSQKHWLSDRT 15 TYQGHTFEDSTKKCADSNPRGV 16 GGHFPP 17 CCVADPETQMTPSSEMF 18CCVADPETQMTPSSEMF 19 CCVTDVQTTNMDVPAGQ 20 TCCVTDIPPPDYEQSLG 21CCESDIPLNELHALADP 22 CCKSDIPSPVTQFNTMK 23 CCQSDVPHQPGINDLHV 24CCMSDTPDISRLPVPDS 25 CCMSDSPADPNRGLPIW 26 CCLSDDAPTLPVRR 27CCITDVPQGVMYKGSPD 28 ECKVDGQLSDSPLLRNN 29 CCMTDDPMDPNSTWAIR 30CCMTDDPMYTNSTWAIR 31 CCVDDTPNSGLAMRVSK 32 CCEVDDFPTHHPGWTLR 33SCNLNHQSCDIPPVKQI 34 CCMADQELDLGHNAANA 35 CCVMDLELASGF 36 CCVMDIEVRGSA37 CCQRDVELVFGS 38 CCRADFEVGNGG 39 CCVSDEPAGVRD 40 GAGWQEKDKELR 41GAMTAGQLSDLP 42 VAGGQVVDRELK 43 KAGEQAMDMELR 44 RGRNQIMDLEI 45QIDRQITDTLL 46 REQQISDVPRV 47 CQAMDAEILNQV 48 GQMMDTELLNR 49 SMEGQVRDIQV50 YQQRDLELLAE 51 SMGQKVDRELV 52 SMGQEVDRELV 53 AENDQMVDWEI 54GGWQESDIPGR 55 GGWQEKDKELR 56 HCCRIDREVSGA 57 DCDWINPPDPPHFWKDT 58DALDERAWRARA 59 RASGKPVNHSTRKEEKQRNGTL 60 GTRDWIEGE 61PHLPRALMRSTTKTSGPRA 62 PEWPGSRDKRT 63 EQKDE 64 LSRPSPFDLFIRKSPTITC 65WLHNEVQLPDARHSTTQPRKT 66 CRASGKPVNHSTRKEEKQRNGLL 67 GKPVNHSTGGC 68GKPVNHSTRKEEKQRNGC 69 CGKPVNHSTRKEEKQRNGLL 70 RASGKPVNHSTGGC 71CGTRDWIEGLL 72 CGTRDWIEGETL 73 GTRDWIEGETGC 74 CHPHLPRALMLL 75CGTHPHLPRALM 76 THPHLPRALMRSC 77 GPHLPRALMRSSSC 78 APEWPGSRDKRTC 79APEWPGSRDKRTLAGGC 80 CGGATPEWPGSRDKRTL 81 CTRKDRSGPWEPA 82 CGAEWEQKDEL83 AEWEQKDEFIC 84 GEQDKEFIC 85 CAEGEQKDEL 86 LFIRKS 87 PSKGTVN 88LHNEVQLPDARHSTTQPRKTKGS 89 SVNPGK 90 CPEWPGCRDKRTG 91 TPEWPGCRDKRCG 92DPEWPGSRDKKGSC 93 DWPGSRDKRKGSC 94 DATPEWPGSRDKRTLKGSC 95

[0030] Accordingly examples of peptides listed in table 1, which havebeen modified to be specific disulphide bridge cyclised peptidessuitable for the present invention are listed in table 2. TABLE 2modified cyclic peptides. Peptide sequence SEQ ID NO. CLEDGQVMDVDLC 96CFINKQMADLELCPRE 97 CFMNKQLADLELCPRE 98 CLEDGQVMDVDLCPREAAEGDK 99CLEDGQVMDVDLCGGSSGGP 100 CLEDGQVMDVDCPREAAEGDK 101 KCREVWLGESETIMDCE 102ACREVWLGESETIMDCD 103 SCREVWLGESETVMDCG 104 NCQDLMLREDAGCWSKM 105DCEEPMCSPVLLQQLKL 106 CFINKQMADLELC 107 CFMNKQLADLELC 108KCREVWLGESETIMDC 109 HCQQVFFPQDYLWCQRG 110 SCREVWLGGSEMIMDCE 111ECNQNLSGSLRHVDLNC 112 DCEEPMCSPVLLQKLKP 113 SCREVWLGGSEMIMDCE 114RCDQQLPRDSYTFCMMS 115 SCPAFPREGDLCAPPTV 116 FCPEPICSPPLSRMTLS 117VCDECVSRELAL 118 WCLEPECAPGLL 119 VCDECVSRELAL 120 DCLSKGQMADLC 121SCQGREVRRECW 122 WCREVWLGESETIMDCE 123 ACREVWLGESETIMDCD 124GCAEPKCWQALHQKLKP 125 ECRGPNMQMQDHCPTTD 126 QCNAVLEGLQMVDHCWN 127HCKNEFKKGQWTYSCSD 128 QCRQFVMNQSEKEFGQC 129 NCFMNKQLADLELCPRE 130SCAYTAQRQCSDVPNPG 131 GCFMNKQMADLELCPRTAA 132 ACFMNKQMADLELCPRVAA 133GCFINKQLADLELCPRVAA 134 GCFMNKQLADWELCPRAAA 135 ECFMNKQLADSELCPRVAA 136GCFMNKQLADPELCPREAE 137 GCFMNKQLVDLELCPRGAA 138 GCFMNKQLADLELCPREAA 139GCFMNKQQADLELCPRGAA 140 GCFINKQMADLELCPREAA 141 CLEDGQVMDVDCPREAAEGD 142CLEDGQVMDVDLCPREAAEGD 143 QCNAVLEGLQMVDHCWN 144 ECLKIEQQCADIVEIPR 145SCAYTAQRQCSDVPNPG 146 ECRGPNMQMQDHCPTTD 147 ECLVYGQMADCAAGGWP 148QCRQFVMNQSEKEFGQC 149 HCKNEFKKGQWTYSCSD 150 CAPGMGCWESVK 151SCREVWLGGSEMIMDCE 152 SCPAFPREGDLCAPPTV 153 FCPEPICSPPLSRMTLS 154ECNQNLSGSLRHVDLNC 155 RCDQQLPRDSYTFCMMS 156 HCQQVFFPQDYLWCQRG 157DCEEPMCSPVLLQKLKP 158 NCQDQMLREDAGCWSKI 159 HCEEPEYSPATRVFCGR 160ACFSRNGQVTDVPHSCY 161 KCPTYPKPNDRCLWPVP 162 YCPKYPLEGDCLLDNDY 163RCEEWLCIPPAPAFAPP 164 TCGQSELCASLETHHV 165 NCNDNPMLDCMPAWSS 166SCQGREVRRECW 167 VCDECVSRELAL 168 WCLEPECAPGLL 169 DCLSKGQMADLC 170VCDECVSRELAL 171 GCPTWPRVGDHC 172 RCQSARVVPECW 173 SCAPSGDCGYKG 174GCPMWPQPDDEC 175 ECPRWPLMGDGC 176 GCQVGELVWCRE 177 QCVRDGTRKVCM 178TCLVDRQESDVC 179 DCVVDGDRLVCL 180 RCEQGALRCVGE 181 VCPPGWKNLGCN 182MCQGWEIVSECW 183 ADGAGCFMNKQMADLELCPREAAEA 184 ADGAGCFMNKQMADLELCPRTAAEA185 ADGAGCFMNKQMADLELCPRVAAEA 186 ADGAGCFINKQLADLELCPRVAAEA 187ADGAGCFINKQMADLELCPREAAEA 188 ADGAGCFMNKQMADLEMCPRDDAEA 189ADGAGCFMNKQLADPELCPREAEEA 190 ADGAGCFMNKQLVDLELCPRGAAEA 191ADGAGCFMNNQLADWELCPRAAAEA 192 ADGAGCFMNKQMADWEMCPRAAAEA 193ADGAGCFMNKQQADLELCPRGAAEA 194 ADGAECFMNKQLADSELCPRVAAEA 195ADGAGCFMNKQLADLELCPREAAEA 196 ADGAGCFINMQMADQELCPRAAAEA 197ADGAGCFINKQMSDFELCPREAGEA 198 ADGAGCFINKQMADLELCTREAAEA 199ADGAGCFINKQMADLELCPRQAAEA 200 ADGAGCFINNQMADLELCPRGGAEA 201ADGAGCFINKQMADWELCPREGAEA 202 ADGAGCFINKQMADLELCPSQAAEA 203ADGAGCFINKQMADLELCPREGAEA 204 ADGAGCFINKQMADSELCPREPAEA 205ADGAGCFIKKQMADLELCPREAWEA 206 ADGAECFINKQMADRELCAREVAEA 207ADGAGCFIDKQMADLELCPRAAAEA 208 ADGAGCFINKQMADLELCRREAGEA 209ADGAGCFKNKQMVDSELCARQAAEA 210 ADGAGCFQNKQMADLELCPREAAEA 211ADGAECFINKQRADLELCPGEAAEA 212 ADGAGCFINKQMADSELCPAAAAEA 213ADGAGCFINRQMADPELCPREAAEA 214 ADGAGCFIEKQMADMELCQARAAEA 215ADGAGCFINKQMADWELCPREAAEA 216 ADGAGCFINNQMADLELCPREAAEA 217ADGAGCFIEKQMADMELCQRETAEA 218 ADGAGCFINKQMADMELCPREAAEA 219ADGAGCFINKQMADLELCPREAAEA 220 ADGAGCFRNKQMADLELCPREAAEA 221ADGAGCFRNKQMADLELCPREAAEA 222 ADGAGCFINRQLADMELCSRGAAEA 223ADGAECFINRQMADLELCGREAAEA 224 ADGAGCFISPQLADWKRCMREAAEA 225ADGAGCSIHTQMADWERCLREGAEA 226 ADGAGCSIHRQMADWERCLREGAEA 227CSSCDGGGHKPPTIQC 228 CLQSSCDGGGHFPPTIQLLC 229 APCWPGSRDCRTLAG 230ACPEWPGSRDRCTLAG 231 CATPEWPGSRDKRTLCG 232 CATPEWPGSRDKRTCG 233TPCWPGSRDKRCG 234 CSRPSPFDLFIRKSPTITC 235 CSRPSPFDLFIRKSPTIC 236CSRPSPFDLFIRKSPTC 237 CSRPSPFDLFIRKSPC 238 CRPSPFDLFIRKSPC 239CRPSPFDLFIRKSPTC 240 CRPSPFDLFIRKSPTIC 241 CRPSPFDLFIRKSPTITC 242CPSPFDLFIRKSPTITC 243 CPSPFDLFIRKSPTIC 244 CPSPFDLFIRKSPTC 245CPSPFDLFIRKSPTC 246 CYAFATPEWPGSRDKRTLAC 247 CYAFATPEWPGSRDKRTLC 248CYAFATPEWPGSRDKRTC 249 CYAFATPEWPGSRDKRC 250 CAFATPEWPGSRDKRC 251CAFATPEWPGSRDKRTC 252 CAFATPEWPGSRDKRTLC 253 CAFATPEWPGSRDKRTLAC 254CFATPEWPGSRDKRTLAC 255 CFATPEWPGSRDKRTLC 256 CFATPEWPGSRDKRTC 257CFATPEWPGSRDKRC 258 CTWSRASGKPVNHSTRC 259 CTWSRASGKPVNHSTC 260CTWSRASGKPVNHSC 261 CTWSRASGKPVNHC 262 CWSRASGKPVNHC 263 CWSRASGKPVNHSC264 CWSRASGKPVNHSTC 265 CWSRASGKPVNHSTRC 266 CSRASGKPVNHSTRC 267CSRASGKPVNHSTC 268 CSRASGKPVNHSC 269 CSRASGKPVNHC 270 CQWLHNEVQLPDARHSC271 CQWLHNEVQLPDARHC 272 CQWLHNEVQLPDARC 273 CQWLHNEVQLPDAC 274CWLHNEVQLPDAC 275 CWLHNEVQLPDARC 276 CWLHNEVQLPDARHC 277CWLHNEVQLPDARHSC 278 CLHNEVQLPDARHSC 279 CLHNEVQLPDARHC 280CLHNEVQLPDARC 281 CLHNEVQLPDAC 282 CPSPFDLFIRKSPCGSK 283CPSPFDLFIRKSPTCGSK 284 FAGCSRASGKPVNHCGAAEG 285 FAGCSRASGKPVNHSCGAAEG286 FAGCSRASGKPVNHSTCGAAEG 287 FAGCSRASGKPVNHSTRCGAAEG 288CSRASGKPVNHCGSK 289 CSRASGKPVNHSCGSK 290 CSRASGKPVNHSTCGSK 291FAGCFATPEWPGSRDKRCGAAEG 292 FAGCFATPEWPGSRDKRTCGAAEG 293FAGCFATPEWPGSRDKRTLCGAAEG 294 FAGCFATPEWPGSRDKRTLACGAAEG 295CPEWPGSRDKRCGSK 296 CWPGSRDKRCGSK 297 CPEWPGSRDKRCGAAEG 298FAGCLHNEVQLPDACGAAEG 299 FAGCLHNEVQLPDARCGAAEG 300FAGCLHNEVQLPDARHCGAAEG 301 FAGCLHNEVQLPDARHSCGAAEG 302FAGCLHNEVQLPDASGAAEG 303 CPEWPGSRDRCGSK 304 CWPGSRDRRCGSK 305CDSNPRGVSAADSNPRGVSC 306 CLVVDLAPSKGTVNC 307 CKQRNGTLC 308 CEEKQRNGTLTVC309 CHPHLPRC 310 CTHPHLPRAC 311 CVTHPHLPRALC 312 CRVTHPHLPRALMC 313CXRVTHPHLPRALMRC 314 CQXRVTHPHLPRALMRSC 315 CYQXRVTHPHLPRALMRSTC 316CPEWPGSRDKRC 317 CRQRNGTLC 318 CEERQRNGTLTVC 319 CMRVTHPHLPRALMRC 320CQMRVTHPHLPRALMRSC 321 CYQMRVTHPHLPRALMRSTC 322 ACPEWPGSRDRCTLAG 323GGCLEDGQVMDVDC 324 CLEDGQVMDCGSK 325 CLEDGQVMDVDLCGSK 326CLEDGQVMDVDLCPREAAEGDK 327 CLEDGQVMDVDLCGGSSGGK 328

[0031] Immunogens produced by the process of the present invention whichmay incorporate the modified peptides of table 1, or the cyclic peptidesof table 2, form a preferred aspect of the present invention. Mimotopeswhich have the same characteristics as these peptides, and immunogenscomprising such mimotopes which generate an immune response whichcross-react with the IgE epitope in the context of the IgE molecule,also form part of the present invention. The meaning of mimotope isdefined as an entity which is sufficiently similar to the native IgEpeptides listed in tables 1 or 2, so as to be capable of beingrecognised by antibodies which recognise the native IgE peptide;(Gheysen, H. M., et al., 1986, Synthetic peptides as antigens. Wiley,Chichester, Ciba foundation symposium 119, p130-149; Gheysen, H. M.,1986, Molecular Immunology, 23,7, 709-715); or are capable of raisingantibodies, when coupled to a suitable carrier, which antibodiescross-react with the native IgE epitope.

[0032] The preferred peptides to be used in the process or inmmunogensof the present invention mimic the surface exposed regions of the IgEstructure, however, within those regions the dominant aspect is thoughtby the present inventors to be those regions within the surface exposedarea which correlate to a loop structure. The structure of the domainsof IgE are described in “Introduction to protein Structure” (page 304,2^(nd) Edition, Branden and Tooze, Garland Publishing, New York, ISBN 08153 2305-0) and take the form a β-barrel made up of two opposinganti-parallel β-sheets (see FIG. 8). The immunogens may comprise adisulphide bridge cyclised peptide which is a sequence derived from aloop of the IgE domains. Preferred examples of this are the A-B loop ofCε3, the A-B loop of Cε4, the C-D loop of Cε3, the C-D loop of Cε4, theA-B loop of Cε2 and the C-D loop of Cε2.

[0033] Peptide mimotopes of the above-identified IgE epitopes may bedesigned for a particular purpose by addition, deletion or substitutionof elected amino acids. Thus, the peptides of the present invention maybe modified for the purposes of ease of conjugation to a proteincarrier. For example, it may be desirable for some chemical conjugationmethods to include a terminal cysteine to the IgE epitope. In additionit may be desirable for peptides conjugated to a protein carrier toinclude a hydrophobic terminus distal from the conjugated terminus ofthe peptide, such that the free unconjugated end of the peptide remainsassociated with the surface of the carrier protein. This reduces theconformational degrees of freedom of the peptide, and thus increases theprobability that the peptide is presented in a conformation which mostclosely resembles that of the IgE peptide as found in the context of thewhole IgE molecule. For example, the peptides may be altered to have anN-terminal cysteine and a C-terminal hydrophobic amidated tail.Alternatively, the addition or substitution of a D-stereoisomer form ofone or more of the amino acids may be performed to create a beneficialderivative, for example to enhance stability of the peptide. Thoseskilled in the art will realise that such modified peptides, ormimotopes, could be a wholly or partly non-peptide mimotope wherein theconstituent residues are not necessarily confined to the 20 naturallyoccurring amino acids. In addition, these may be cyclised by techniquesknown in the art to constrain the peptide into a conformation thatclosely resembles its shape when the peptide sequence is in the contextof the whole IgE molecule. A preferred method of cyclising a peptidecomprises the addition of a pair of cysteine residues to allow theformation of a disulphide bridge.

[0034] Further, those skilled in the art will realise that mimotopes orimmunogens of the present invention may be larger than theabove-identified epitopes, and as such may comprise the sequencesdisclosed herein. Accordingly, the mimotopes of the present inventionmay consist of addition of N and/or C terminal extensions of a number ofother natural residues at one or both ends. The peptide mimotopes mayalso be retro sequences of the natural IgE sequences, in that thesequence orientation is reversed; or alternatively the sequences may beentirely or at least in part comprised of D-stereo isomer amino acids(inverso sequences).

[0035] Also, the peptide sequences may be retro-inverso in character, inthat the sequence orientation is reversed and the amino acids are of theD-stereoisomer form. Such retro or retro-inverso peptides have theadvantage of being non-self, and as such may overcome problems ofself-tolerance in the immune system (for example P14c).

[0036] Alternatively, peptide mimotopes may be identified usingantibodies which are capable themselves of binding to the IgE epitopesof the present invention using techniques such as phage displaytechnology (EP 0 552 267 B1). This technique, generates a large numberof peptide sequences which mimic the structure of the native peptidesand are, therefore, capable of binding to anti-native peptideantibodies, but may not necessarily themselves share significantsequence homology to the native IgE peptide. This approach may havesignificant advantages by allowing the possibility of identifying apeptide with enhanced immunogenic properties (such as higher affinitybinding characteristics to the IgE receptors or anti-IgE antibodies, orbeing capable of inducing polyclonal immune response which binds to IgEwith higher affinity), or may overcome any potential self-antigentolerance problems which may be associated with the use of the nativepeptide sequence. Additionally this technique allows the identificationof a recognition pattern for each native-peptide in terms of its sharedchemical properties amongst recognised mimotope sequences.

[0037] Alternatively, peptide mimotopes may be generated with theobjective of increasing the immunogenicity of the peptide by increasingits affinity to the anti-IgE peptide polyclonal antibody, the effect ofwhich may be measured by techniques known in the art such as (Biocoreexperiments). In order to achieve this the peptide sequence may beelectively changed following the general rules:

[0038] To maintain the structural constraints, prolines and glycinesshould not be replaced

[0039] Other positions can be substituted by an amino acid that hassimilar physicochemical properties.

[0040] As such, each amino acid residue can be replaced by the aminoacid that most closely resembles that amino acid. For example, A may besubstituted by V, L or I, as described in the following table 3.Exemplary Preferred Original residue substitutions substitution A V, L,I V R K, Q, N K N Q, H, K, R Q D E E C S S Q N N E D D G A A H N, Q, K,R N I L, V, M, A, F L L I, V, M, A, F I K R, Q, N R M L, F, I L F L, V,I, A, Y, W W P A A S T T T S S W Y, F Y Y W, F, T, S F V I, L, M, F, A L

[0041] The present invention, therefore, provides a process for themanufacture of a vaccine and novel immunogens comprising disulphidebridge cyclised peptides conjugated by the process of the presentinvention, and the use of the immunogens in the manufacture ofpharmaceutical compositions for the prophylaxis or therapy of disease.Preferably the process and the immunogens of the present invention areused in vaccines for the immunoprophylaxis or therapy of allergies.

[0042] It is envisaged that the peptides used in the process of presentinvention will be of a small size. Peptides, therefore, should be lessthan 100 amino acids in length, preferably shorter than 75 amino acids,more preferably less than 50 amino acids, and most preferable within therange of 4 to 25 amino acids long.

[0043] The most preferred peptides for use in the processes andconjugates of the present invention are SEQ ID NO.s 99, 304, 305, 306,307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,321, 322, 323, 324, 325, 326, 327, and 328.

[0044] The types of immunogenic carriers used in the immunogens of thepresent invention will be readily known to the man skilled in the art.The preferred function of the carrier is to provide cytokine help inorder to help induce an immune response against the IgE peptide. Anon-exhaustive list of carriers which may be used in the presentinvention include: Keyhole limpet Haemocyanin (KLH), serum albumins suchas bovine serum albumin (BSA), inactivated bacterial toxins such astetanus or diptheria toxins (TT and DT), or recombinant fragmentsthereof (for example, Domain 1 of Fragment C of TT, or the translocationdomain of DT), or the purified protein derivative of tuberculin (PPD).Alternatively, the process may be used to conjugate the cyclic peptidesdirectly to liposome carriers, which may additionally comprise carrierscapable of providing T-cell help. Preferably the ratio of peptides tocarrier is in the order of 1:1 to 20:1, and preferably each carriershould carry between 3-15 peptides.

[0045] In an embodiment of the invention a preferred carrier is ProteinD from Haemophilus influenzae (EP 0 594 610 B1). Protein D is anIgD-binding protein from Haemophilus influenzae and has been patented byForsgren (WO 91/18926, granted EP 0 594610 B1). In some circumstances,for example in recombinant immunogen expression systems it may bedesirable to use fragments of protein D, for example Protein D 1/3^(rd)(comprising the N-terminal 100-110 amino acids of protein D (GB9717953.5)).

[0046] Peptides can be readily prepared using the ‘Fmoc’ procedure,utilising either polyamide or polyethyleneglycol-polystyrene (PEG-PS)supports in a fully automated apparatus, through techniques well knownin the art (techniques and procedures for solid phase synthesis aredescribed in ‘Solid Phase Peptide Synthesis: A Practical Approach’ by E.Atherton and R. C. Sheppard, published by IRL at Oxford University Press(1989)) followed by acid mediated cleavage to leave the linear,deprotected, modified peptide. This peptide can be readily oxidised andpurified to yield the disulphide-bridge modified peptide, usingmethodology outlined in ‘Methods in Molecular Biology, Vol. 35: PeptideSynthesis Protocols (ed. M. W. Pennington and B. M. Dunn), chapter 7,pp9l-171 by D. Andreau et al.

[0047] Alternatively, the peptides may be produced by recombinantmethods, including expressing nucleic acid molecules encoding themimotopes in a bacterial or mammalian cell line, followed bypurification of the expressed mimotope. Techniques for recombinantexpression of peptides and proteins are known in the art, and aredescribed in Maniatis, T., Fritsch, E. F. and Sambrook et al., Molecularcloning, a laboratory manual, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989).

[0048] The amount of protein in each vaccine dose is selected as anamount which induces an immunoprotective response without significantadverse side effects in typical vaccines. Such amount will varydepending upon which specific immunogen is employed and how it ispresented. Generally, it is expected that each dose will comprise 1-1000μg of protein, preferably 1-500 μg, more preferably 1-100 μg, of which 1to 50 μg is the most preferable range. An optimal amount for aparticular vaccine can be ascertained by standard studies involvingobservation of appropriate immune responses in subjects. Following aninitial vaccination, subjects may receive one or several boosterimmunisations adequately spaced.

[0049] Vaccines of the present invention, may advantageously alsoinclude an adjuvant. Suitable adjuvants for vaccines of the presentinvention comprise those adjuvants that are capable of enhancing theantibody responses against the immunogen. Adjuvants are well known inthe art (Vaccine Design—The Subunit and Adjuvant Approach, 1995,Pharmaceutical Biotechnology, Volume 6, Eds. Powell, M. F., and Newman,M. J., Plenum Press, New York and London, ISBN 0-306-44867-X). Preferredadjuvants for use with immunogens of the present invention includealuminium or calcium salts (for example hydroxide or phosphate salts).Preferred adjuvants for use with immunogens of the present inventioninclude: aluminium or calcium salts (hydroxide or phosphate), oil inwater emulsions (WO 95/17210, EP 0 399 843), or particulate carrierssuch as liposomes (WO 96/33739). Immunologically active saponinfractions (e.g. Quil A) having adjuvant activity derived from the barkof the South American tree Quillaja Saponaria Molina are particularlypreferred. Derivatives of Quil A, for example QS21 (an HPLC purifiedfraction derivative of Quil A), and the method of its production isdisclosed in U.S. Pat. No.5,057,540. Amongst QS21 (known as QA21) otherfractions such as QA17 are also disclosed. 3 De-O-acylatedmonophosphoryl lipid A is a well known adjuvant manufactured by RibiImmunochem, Montana. It can be prepared by the methods taught in GB2122204B. A preferred form of 3 De-O-acylated monophosphoryl lipid A isin the form of an emulsion having a small particle size less than 0.2cmin diameter (EP 0 689 454 B1).

[0050] Adjuvants also include, but are not limited to, muramyl dipeptideand saponins such as Quil A, bacterial lipopolysaccharides such as3D-MPL (3-O-deacylated monophosphoryl lipid A), or TDM. As a furtherexemplary alternative, the protein can be encapsulated withinmicroparticles such as liposomes, or in non-particulate suspensions ofpolyoxyethylene ether (UK Patent Application No. 9807805.8).Particularly preferred adjuvants are combinations of 3D-MPL and QS21 (EP0 671 948 B1), oil in water emulsions comprising 3D-MPL and QS21 (WO95/17210, PCT/EP98/05714), 3D-MPL formulated with other carriers (EP 0689 454 B1), or QS21 formulated in cholesterol containing liposomes (WO96/33739), or immunostimulatory oligonucleotides (WO 96/02555).Alternative adjuvants include those described in WO 99/52549.

[0051] The vaccines of the present invention will be generallyadministered for both priming and boosting doses. It is expected thatthe boosting doses will be adequately spaced, or preferably given yearlyor at such times where the levels of circulating antibody fall below adesired level. Boosting doses may consist of the peptide in the absenceof the original carrier molecule. Such booster constructs may comprisean alternative carrier or may be in the absence of any carrier.

[0052] In a further aspect of the present invention there is provided animmunogen or vaccine as herein described for use in medicine.

[0053] Preferably, the vaccine preparation of the present invention maybe used to protect or treat a mammal susceptible to, or suffering fromallergies, by means of administering said vaccine via systemic ormucosal route. These administrations may include injection via theintramuscular, intraperitoneal, intradermal or subcutaneous routes; orvia mucosal administration to the oral/alimentary, respiratory,genitourinary tracts. A preferred route of administration is via thetransdermal route, for example by skin patches. Accordingly, there isprovided a method for the treatment of allergy, comprising theadministration of a peptide, immunogen, or ligand of the presentinvention to a patient who is suffering from or is susceptible toallergy.

[0054] Vaccine preparation is generally described in New Trends andDevelopments in Vaccines, edited by Voller et al., University ParkPress, Baltimore, Md., U.S.A. 1978. Conjugation of proteins tomacromolecules is disclosed by Likhite, U.S. Pat. No. 4,372,945 and byArmor et al., U.S. Pat. No. 4,474,757.

[0055] The present invention is illustrated by but not limited to thefollowing examples.

EXAMPLE 1

[0056] Conjugation of Disulphide Cyclisedpeptide to a Carries; byConjugating a Maleimide Activated Peptide to Thiolated Protein D or BSAas a Carrier.

[0057] In the present example, a maleimide derivatised cyclic peptide isreacted with a thiol bearing carrier. The thiol group being generated oneither Protein D (PD) or BSA as the carrier by reduction of the SPDPderivative of the carrier.

[0058] N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) is aheterobifunctional cross-linking agent which under mild conditions,reacts by its NHS-ester group with amino groups of the protein (FIG. 3)(Hermanson G. T. Bioconjugate Techniques, 1996). NHS-ester crosslinkingreactions are most commonly performed in phosphate,bicarbonate/carbonate and borate buffers. Other buffers can be usedprovided they do not contain primary amines. Treatment of a SPDPmodified protein with DTT (Dithiothreitol, or another disulfide-reducingagent) releases the pyridine-2-thione leaving group and forms a freesulfhydryl (FIG. 3A). The reaction is generally performed with 25 mM DTTat pH 4.5 to avoid the reduction of the protein's S—S bonds. For proteinnot containing S—S bonds, the DTT reduction may be performed at pH 7-9.The reaction between a maleimide group added on the peptide and thesulfhydryl groups present on the carrier produces the immunogen of thepresent invention (FIG. 3B). The maleimide-activated peptide wasobtained by reaction between the peptide (P) and a heterobifunctionnalcross-linking reagent like GMBS (gamma-maleimidobutyric acidN-hydroxysuccinimide ester).

[0059] Methods

[0060] SPDP Modified Protein

[0061] BSA (Pierce) is dissolved at a concentration of 10 mg/ml in 50 mMsodium phosphate, 0.15 M NaCl, pH 7.2. SPDP was dissolved at aconcentration of 6.2 mg/ml in DMSO (makes a 20 mM stock solution). Asufficient quantity of the stock solution of SPDP was then added to theprotein to be modified (for BSA, a 15 fold molar excess of SPDP overprotein, and for PD, a 25 fold molar excess). After one hour at roomtemperature, the modified protein was purified from reaction by productsby dialysis against 50 mM sodium phosphate, 10 mM EDTA pH 6.8 or by gelfiltration. The sample is applied on a desalting column (Sephadex G25)equilibrated with phosphate buffer pH 6.8 (or 100 mM sodium acetate,0.15 M NaCl, 1 mM EDTA pH 4.5 if S-S containing proteins are to bereduced in the next step). Fractions of 1 ml are collected and monitoredby adsorbance at 280 nm. Fractions containing SPDP modified protein arepooled.

[0062] The number of thiopyridyl groups introduced in BSA is estimatedspectrophotometrically: transfer 200 μl of modified BSA in aspectrophotometer cuvette and add 200 μl of 50 mM mercaptoethanol in 100mM phosphate buffer, pH 7. Measure absorbance at 343 nm before and afteraddition of mercaptoethanol. Evaluate the quantity of thiopyridoneliberated using A_(343 nm)=8000 M⁻¹cm⁻¹.

[0063] Use of DTT to Cleave Disulfide-Containing Cross-Linking Agents

[0064] DTT was added to a final concentration of 1-10 mM. Incubate for 2h at room temperature. For removal of excess of DTT, gel filtrationusing Sephadex G-25 was used. To maintain the stability of the exposedsulfhydryl groups, 10 mM EDTA was included in the chromatography buffer(100 mM sodium phosphate pH 6.8). The presence of oxidized DTT can bemonitored during elution by measuring the absorbance at 280 nm.

[0065] Maleimide Modified Peptide

[0066] Peptide was dissolved in 100 mM sodium phosphate pH 6.8. GMBS(Pierce) was then added to the peptide sample. A 2.5-fold molar excessof the cross-linker over the peptide was used. After 1 hr at roomtemperature, reaction by-products were removed by gel filtration using asephadex G-10 (100 mM sodium phosphate pH 6.8). Fractions of 1 ml werecollected and monitored by adsorbance at 280 nm. Presence of maleimidegroup was demonstrated by Ellman's reaction.

[0067] Reaction Between SPDP Modified Protein and Maleimide ActivatedPeptide

[0068] An excess of maleimide activated peptide (about 22 fold molarexcess of maleimide activated peptide over the protein) was added to theSPDP modified protein and was agitated during 1 hr at room temperaturefollowed by three dialysis against 100 mM Na phosphate pH 6.8. Afterfiltration through 0.2 μm pore size (millipore filter), protein contentwas estimated by Lowry.

[0069] Results

[0070] 1. Obtention of the SPDP Modified Protein

[0071] Several assays were conducted with different concentrations ofSPDP using BSA or PD as carrier.

[0072] 1 .a Assays on PD

[0073] The number of thiopyridyl groups introduced was estimatedspectrophotometrically by evaluation of thiopyridone liberated afteraddition of mercaptoethanol. Several assays were realized using PD at aconcentration of 6.6 mg/ml or 10 mg/ml. results At least 14 thiopyridylgroups could be introduced on PD (FIG. 4). However, at a concentrationof 10 mg/ml of PD only 4-5 thiopyridyl groups could be introduced on PD(FIG. 5). Indeed, precipitation of PD was observed when assays to obtainmore thiopyridyl groups were carried out. However, this precipitation ispartially induced by DMSO used to dissolve SPDP (6.2 mg/ml). Thisproblem could be resolved by using the water-soluble sulfo-LC-SPDP(Sulfosuccinimidyl 6-[2-pyridyldithio)-propionamido]hexanoate).

[0074] 1.b Assays on BSA

[0075] A maximum of 8 to 10 thiopyridyl groups can be added on BSA. Ahigher thiopyridyl number can be obtained if a 20 fold molar excess ofSPDP over BSA was used (FIG. 6). However, a slight clouding was thenobserved during the reaction resulting in a lower yield of SPDP modifiedBSA.

[0076] Assays of reduction of pyridyl disulfide with DTT were carriedout in sodium acetate pH 4.5 (to avoid reduction of native disulphidebonds) or in phosphate buffer (for SPDP modified PD). Efficacy of DTTwas determined by release of pyridine-2-thione.

[0077] 2. Conjugation of Constrained p15 Peptides

[0078] Five constrained peptides were conjugated to the BSA using thechemistry described hereabove: Original sequence: EDGQVMDVD (SEQ IDNO. 1) p15a: GGCLEDGQVMDVDC (SEQ ID NO. 324) p15b: Ac-CLEDGQVMDCGSK-NH₂(SEQ ID NO. 325) p15c: Ac-CLEDGQVMDVDLCGSK-NH₂ (SEQ ID NO. 326) p15d:Ac-CLEDGQVMDVDLCPREAAEGDK-NH₂ (SEQ ID NO. 327) p15e:Ac-CLEDGQVMDVDLCGGSSGGK-NH₂ (SEQ ID NO. 328)

[0079] The resulting conjugates were soluble and were characterized bySDS-PAGE (Coomassie blue-staining) (FIG. 7).

[0080] 3. Conjugation of Constrained p14 Peptides

[0081] Three constrained peptides were conjugated: Original sequence:PEWPGSRDKRT (SEQ ID NO. 63) p14e: ACPEWPGSRDRCTLAG-NH₂ (SEQ ID NO. 323)p14f: Ac-CPEWPGSRDRCGSK-NH₂ (SEQ ID NO. 304) p14i: Ac-CWPGSRDRRCGSK-NH₂(SEQ ID NO. 305)

[0082] The resulting conjugates were soluble and were characterized bySDS-PAGE (coomassie blue-staining and western blot) (FIG. 7B, lane 7,FIG. 8 and FIG. 9).

[0083] 4. Thiol-Disulfide Exchange

[0084] Compounds containing a disulfide group are able to participate indisulfide exchange reactions with another thiol. The disulfide exchangeprocess involves attack of the thiol at the disulfide, breaking the S—Sbond, with subsequent formation of a new mixed disulfide constituting aportion of the original disulfide compound. If the thiol is present inexcess, the mixed disulfide can go on to form a symmetrical disulfideconsisting entirely of the thiol reducing agent. If the thiol is notpresent in large excess, the mixed disulfide product is the end result.

[0085] In order to test if a disulfide interchange could be observedduring the reaction between BSA-SH and the maleimide activated disulfidebridge cyclised peptide, a reaction between BSA-SH and the unmodifiedp14i peptide was realized in the same coupling conditions (buffer, pH,ratio peptide/carrier and temperature). After 1 hour, the sample wasdialyzed or applied on a desalting column (sephadex G25) equilibratedwith phosphate buffer pH 6.8. The resulting product was analyzed onSDS-PAGE (coomassie blue staining) (FIG. 10). A positive control wasincluded resulting from the reaction between SPDP-modified BSA and p14apeptide (AcAPEWPGSRDKRTLAGGC) in which disulfide interchange occurs(FIG. 3A). The resulting conjugate was purified by dialysis or by gelfiltration.

[0086] No increase of the molecular size was seen for the productresulting of the reaction between BSA-SH and p14i (FIG. 10A: Lane 9).Moreover, no protein was detected with the mAb 31 (FIG. 1B: lane 9)suggesting the absence of disulfide interchange during the reaction atleast in the conditions used for the coupling.

[0087] Conclusions

[0088] The combination of two chemistries was used to conjugateconstrained peptides to a carrier. Soluble conjugates with 6 to 8peptides on the carrier were obtained and were characterized by SDS-PAGEwith antibodies against p14. The resulting conjugates were principallyobtained by the reaction between the GMBS activated peptide and BSA-SHand not by disulfide interchange as confirmed by Western-blot. Theseresults demonstrate that these chemistries can be used to conjugateconstrained peptides to a carrier. In the above examples the maleimidewas added to the peptide via reaction of maleimide-N-hydroxysuccinimideester reagents with a lysine side-chain or with a N-terminal aminogroup. It is clear that alternative methods of adding the maleimidegroup can be readily conceived: notably for peptides containing a lysinewithin the epitope, the maleimide can be added during peptide synthesisprior to final deprotection of the side-chains and cleavage of thepeptide.

EXAMPLE 2

[0089] Immune Response Induced by Different Disulphide BridgedPeptide-BSA Conjugates.

[0090] To evaluate the immunogenicity of the conjugates produced inExample 1, 10 mice per group were immunised intramuscularly (IM) on days0, 14 and 28 with 25 μg of conjugate mixed with AS2 adjuvant (oil/wateremulsion, 3D-MPL, QS21). The serologic response for the P14 peptides wasanalysed by ELISA on days 28 and 42 (14 post III). The results are shownbelow in Table 4. TABLE 4 IgG response against P14 peptides, day 14 postIII Peptide conjugate IgG anti-peptide responses (midpoint titre)Average. st. deviation. geomean P14e 3151 3224 3051 2873 4647 1461 42273821 2345 3200 963 3051 P14f 67086 36031 74838 56496 51304 92885 92868113041 89155 101502 77521 24519 73541 P14I 85882 39268 39460 57276 5083454664 62263 36621 26202 28989 48146 17926 45336

[0091] Immune Response Induced by Different P15-BSA Conjugates.

[0092] The P15 peptide conjugates produced in Example 1 were also usedto immunise 10 mice per group,intramuscularly (IM) on days 0, 14 and 28with 25 μg of conjugate mixed with AS2 adjuvant (oil/water emulsion,3D-MPL, QS21). Anti peptide and anti-IgE antibody responses are shown inTable 5 (14 days post III). Very homogenous responses were obtained withall cyclic P15 peptides. Anti-IgE antibody responses were assayed bycomparison with a monoclonal antibody, mAb 11, which is known torecognise the P15 target site (c-d loop of Cε2) and inhibit histaminerelease in the Human Basophil Assay, the levels of anti-IgE weresubsequently expressed as μg/ml mAb11 equivalents. TABLE 5 Immuneresponse by cyclic P15-BSA conjugates. anti-IgE (μg/ml or BSAanti-peptide (midpoint titre) mAB11 equivalent) conjugate average StDev. geomean average St Dev. geomean P15b 11169 10766 8385 70 104 35P15c 66452 10917 65685 200 64 189 P15d 35118 11601 32801 174 168 111P15e 57432 16589 55207 129 68 113

[0093] Human Basophil Assays

[0094] Two types of assay were performed with human basophils (HBA), oneto determine the anaphylactogenicity of the vaccine induced antibodies,consisting of adding the antibodies to isolated PBMC; and a second tomeasure the inhibition of Lol P I (a strong allergen) triggeredhistamine release by pre-incubation of the HBA with the vaccine inducedantibodies.

[0095] Blood was collected by venepuncture from 4 allergic donors intotubes containing 0.1 volumes 2.7% EDTA, pH 7.0. It is then diluted 1/2with an equal volume of HBH medium containing 0.1% human serum albumin(HBH/HSA). The resulting cell suspension was layered over 50% volumeFicoll-Paque and centrifuged at 400 g for 30 minutes at roomtemperature. The peripheral blood mononuclear cell (PBMC) layer at theinterface is collected and the pellet is discarded. The cells are washedonce in HBH/HSA, counted, and re-suspended in HBH/HSA at a cell densityof 2.0×10⁶ per ml. 100 μl cell suspension are added to wells of aV-bottom 96-well plate containing 100 μl diluted test sample or vaccineinduced antibody. Each test sample is tested at a range of dilutionswith 6 wells for each dilution. Well contents are mixed briefly using aplate shaker, before incubation at 37° C. for 30 minutes with shaking at120 rpm.

[0096] For each serum dilution 3 wells are triggered by addition of 10μl Lol p I extract (final dilution 1/10000) and 3 wells have 10 μlHBH/HSA added for assessment of anaphylactogenicity. Well contents areagain mixed briefly using a plate shaker, before incubation at 37° C.for a further 30 minutes with shaking at 120 rpm. Incubations areterminated by centrifugation at 500 g for 5 min. Supernatants areremoved for histamine assay using a commercially available histamine EIAmeasuring kit (Immunotech). Control wells containing cells without testsample are routinely included to determine spontaneous and triggeredrelease. Wells containing cells ±0.05% Igepal detergent are alsoincluded to determine total cell histarnine.

[0097] The results are expressed as following:

[0098] Anaphylactogenesis Assay

Histamine release due to test samples=% histamine release from testsample treated cells−% spontaneous histamine release.

[0099] Blocking Assay

[0100] The degree of inhibition of histamine release can be calculatedusing the formula:

% inhibition=1−(histamine release from test sample treatedcells*)×100(histamine release from antigen stimulated cells*)

[0101] Values corrected for spontaneous release.

[0102] Results

[0103] The results of the histamine release activity of the P15disulphide bridge cyclised peptides conjugated to the BSA carriers usingthe chemistry of the present invention are shown in FIGS. 11 to 14.

[0104]FIGS. 11, A and B, show the histamine release blocking activity ofantiserum induced by P15c, P15d and P15e; in comparison with thepositive controls: 1079 BSA, PT11 and mAb005, and the negative controlsBSA-BAL (activated carrier alone), anti-BSA, non-specific isotypecontrols (IgG1 and IgG2b); also shown are the data produced forspontanteous release of histamine, and histamine release aftertriggering with allergen, and total histamine content of the cells(released by detergent).

[0105]FIGS. 12, A and B, show the histamine release blocking activity ofantiserum induced by P15c compared to the same controls as in FIG. 11,with the addition of a further positive control 1079 HBC, and oneadditional negative control HBC wt.

[0106]FIG. 13 shows the anaphylactogenicity of the same test samples(antiserum added to HBA in the absence of allergen) as described forFIG. 11 (P15c, P15d and P15e). FIG. 14 shows the anaphylactogenicity ofthe same test samples as described for FIG. 12.

[0107] In summary, P15c, P15d and P15e induced antisera that inhibitedhistamine release from human basophils after triggering with allergen,without the antiserum being anaphylactogenic themselves.

1 328 1 9 PRT Homo sapiens 1 Glu Asp Gly Gln Val Met Asp Val Asp 1 5 2 8PRT Homo sapiens 2 Ser Thr Thr Gln Glu Gly Glu Leu 1 5 3 10 PRT Homosapiens 3 Ser Gln Lys His Trp Leu Ser Asp Arg Thr 1 5 10 4 10 PRT Homosapiens 4 Gly His Thr Phe Glu Asp Ser Thr Lys Lys 1 5 10 5 8 PRT Homosapiens 5 Gly Gly Gly His Phe Pro Pro Thr 1 5 6 6 PRT Homo sapiens 6 ProGly Thr Ile Asn Ile 1 5 7 5 PRT Homo sapiens 7 Phe Thr Pro Pro Thr 1 5 813 PRT Homo sapiens 8 Cys Leu Glu Asp Gly Gln Val Met Asp Val Asp LeuLeu 1 5 10 9 13 PRT Homo sapiens 9 Leu Leu Asp Val Asp Met Val Gln GlyAsp Glu Leu Cys 1 5 10 10 13 PRT Homo sapiens 10 Trp Leu Glu Asp Gly GlnVal Met Asp Val Asp Leu Cys 1 5 10 11 7 PRT Homo sapiens 11 Gln Val MetAsp Val Asp Leu 1 5 12 10 PRT Homo sapiens 12 Leu Glu Asp Gly Gln ValMet Asp Val Asp 1 5 10 13 10 PRT Homo sapiens 13 Cys Ser Thr Thr Gln GluGly Glu Leu Ala 1 5 10 14 6 PRT Homo sapiens 14 Thr Thr Gln Glu Gly Glu1 5 15 11 PRT Homo sapiens 15 Cys Ser Gln Lys His Trp Leu Ser Asp ArgThr 1 5 10 16 22 PRT Homo sapiens 16 Thr Tyr Gln Gly His Thr Phe Glu AspSer Thr Lys Lys Cys Ala Asp 1 5 10 15 Ser Asn Pro Arg Gly Val 20 17 6PRT Homo sapiens 17 Gly Gly His Phe Pro Pro 1 5 18 17 PRT Homo sapiens18 Cys Cys Val Ala Asp Pro Glu Thr Gln Met Thr Pro Ser Ser Glu Met 1 510 15 Phe 19 17 PRT Homo sapiens 19 Cys Cys Val Ala Asp Pro Glu Thr GlnMet Thr Pro Ser Ser Glu Met 1 5 10 15 Phe 20 17 PRT Homo sapiens 20 CysCys Val Thr Asp Val Gln Thr Thr Asn Met Asp Val Pro Ala Gly 1 5 10 15Gln 21 17 PRT Homo sapiens 21 Thr Cys Cys Val Thr Asp Ile Pro Pro ProAsp Tyr Glu Gln Ser Leu 1 5 10 15 Gly 22 17 PRT Homo sapiens 22 Cys CysGlu Ser Asp Ile Pro Leu Asn Glu Leu His Ala Leu Ala Asp 1 5 10 15 Pro 2317 PRT Homo sapiens 23 Cys Cys Lys Ser Asp Ile Pro Ser Pro Val Thr GlnPhe Asn Thr Met 1 5 10 15 Lys 24 17 PRT Homo sapiens 24 Cys Cys Gln SerAsp Val Pro His Gln Pro Gly Ile Asn Asp Leu His 1 5 10 15 Val 25 17 PRTHomo sapiens 25 Cys Cys Met Ser Asp Thr Pro Asp Ile Ser Arg Leu Pro ValPro Asp 1 5 10 15 Ser 26 17 PRT Homo sapiens 26 Cys Cys Met Ser Asp SerPro Ala Asp Pro Asn Arg Gly Leu Pro Ile 1 5 10 15 Trp 27 14 PRT Homosapiens 27 Cys Cys Leu Ser Asp Asp Ala Pro Thr Leu Pro Val Arg Arg 1 510 28 17 PRT Homo sapiens 28 Cys Cys Ile Thr Asp Val Pro Gln Gly Val MetTyr Lys Gly Ser Pro 1 5 10 15 Asp 29 17 PRT Homo sapiens 29 Glu Cys LysVal Asp Gly Gln Leu Ser Asp Ser Pro Leu Leu Arg Asn 1 5 10 15 Asn 30 17PRT Homo sapiens 30 Cys Cys Met Thr Asp Asp Pro Met Asp Pro Asn Ser ThrTrp Ala Ile 1 5 10 15 Arg 31 17 PRT Homo sapiens 31 Cys Cys Met Thr AspAsp Pro Met Tyr Thr Asn Ser Thr Trp Ala Ile 1 5 10 15 Arg 32 17 PRT Homosapiens 32 Cys Cys Val Asp Asp Thr Pro Asn Ser Gly Leu Ala Met Arg ValSer 1 5 10 15 Lys 33 17 PRT Homo sapiens 33 Cys Cys Glu Val Asp Asp PhePro Thr His His Pro Gly Trp Thr Leu 1 5 10 15 Arg 34 17 PRT Homo sapiens34 Ser Cys Asn Leu Asn His Gln Ser Cys Asp Ile Pro Pro Val Lys Gln 1 510 15 Ile 35 17 PRT Homo sapiens 35 Cys Cys Met Ala Asp Gln Glu Leu AspLeu Gly His Asn Ala Ala Asn 1 5 10 15 Ala 36 12 PRT Homo sapiens 36 CysCys Val Met Asp Leu Glu Leu Ala Ser Gly Phe 1 5 10 37 12 PRT Homosapiens 37 Cys Cys Val Met Asp Ile Glu Val Arg Gly Ser Ala 1 5 10 38 12PRT Homo sapiens 38 Cys Cys Gln Arg Asp Val Glu Leu Val Phe Gly Ser 1 510 39 12 PRT Homo sapiens 39 Cys Cys Arg Ala Asp Phe Glu Val Gly Asn GlyGly 1 5 10 40 12 PRT Homo sapiens 40 Cys Cys Val Ser Asp Glu Pro Ala GlyVal Arg Asp 1 5 10 41 12 PRT Homo sapiens 41 Gly Ala Gly Trp Gln Glu LysAsp Lys Glu Leu Arg 1 5 10 42 12 PRT Homo sapiens 42 Gly Ala Met Thr AlaGly Gln Leu Ser Asp Leu Pro 1 5 10 43 12 PRT Homo sapiens 43 Val Ala GlyGly Gln Val Val Asp Arg Glu Leu Lys 1 5 10 44 12 PRT Homo sapiens 44 LysAla Gly Glu Gln Ala Met Asp Met Glu Leu Arg 1 5 10 45 11 PRT Homosapiens 45 Arg Gly Arg Asn Gln Ile Met Asp Leu Glu Ile 1 5 10 46 11 PRTHomo sapiens 46 Gln Ile Asp Arg Gln Ile Thr Asp Thr Leu Leu 1 5 10 47 11PRT Homo sapiens 47 Arg Glu Gln Gln Ile Ser Asp Val Pro Arg Val 1 5 1048 12 PRT Homo sapiens 48 Cys Gln Ala Met Asp Ala Glu Ile Leu Asn GlnVal 1 5 10 49 11 PRT Homo sapiens 49 Gly Gln Met Met Asp Thr Glu Leu LeuAsn Arg 1 5 10 50 11 PRT Homo sapiens 50 Ser Met Glu Gly Gln Val Arg AspIle Gln Val 1 5 10 51 11 PRT Homo sapiens 51 Tyr Gln Gln Arg Asp Leu GluLeu Leu Ala Glu 1 5 10 52 11 PRT Homo sapiens 52 Ser Met Gly Gln Lys ValAsp Arg Glu Leu Val 1 5 10 53 11 PRT Homo sapiens 53 Ser Met Gly Gln GluVal Asp Arg Glu Leu Val 1 5 10 54 11 PRT Homo sapiens 54 Ala Glu Asn AspGln Met Val Asp Trp Glu Ile 1 5 10 55 11 PRT Homo sapiens 55 Gly Gly TrpGln Glu Ser Asp Ile Pro Gly Arg 1 5 10 56 11 PRT Homo sapiens 56 Gly GlyTrp Gln Glu Lys Asp Lys Glu Leu Arg 1 5 10 57 12 PRT Homo sapiens 57 HisCys Cys Arg Ile Asp Arg Glu Val Ser Gly Ala 1 5 10 58 17 PRT Homosapiens 58 Asp Cys Asp Trp Ile Asn Pro Pro Asp Pro Pro His Phe Trp LysAsp 1 5 10 15 Thr 59 12 PRT Homo sapiens 59 Asp Ala Leu Asp Glu Arg AlaTrp Arg Ala Arg Ala 1 5 10 60 22 PRT Homo sapiens 60 Arg Ala Ser Gly LysPro Val Asn His Ser Thr Arg Lys Glu Glu Lys 1 5 10 15 Gln Arg Asn GlyThr Leu 20 61 9 PRT Homo sapiens 61 Gly Thr Arg Asp Trp Ile Glu Gly Glu1 5 62 19 PRT Homo sapiens 62 Pro His Leu Pro Arg Ala Leu Met Arg SerThr Thr Lys Thr Ser Gly 1 5 10 15 Pro Arg Ala 63 11 PRT Homo sapiens 63Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr 1 5 10 64 5 PRT Homo sapiens64 Glu Gln Lys Asp Glu 1 5 65 19 PRT Homo sapiens 65 Leu Ser Arg Pro SerPro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr 1 5 10 15 Ile Thr Cys 66 21PRT Homo sapiens 66 Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg HisSer Thr Thr 1 5 10 15 Gln Pro Arg Lys Thr 20 67 23 PRT Homo sapiens 67Cys Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu 1 5 1015 Lys Gln Arg Asn Gly Leu Leu 20 68 11 PRT Homo sapiens 68 Gly Lys ProVal Asn His Ser Thr Gly Gly Cys 1 5 10 69 18 PRT Homo sapiens 69 Gly LysPro Val Asn His Ser Thr Arg Lys Glu Glu Lys Gln Arg Asn 1 5 10 15 GlyCys 70 20 PRT Homo sapiens 70 Cys Gly Lys Pro Val Asn His Ser Thr ArgLys Glu Glu Lys Gln Arg 1 5 10 15 Asn Gly Leu Leu 20 71 14 PRT Homosapiens 71 Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Gly Gly Cys 1 510 72 11 PRT Homo sapiens 72 Cys Gly Thr Arg Asp Trp Ile Glu Gly Leu Leu1 5 10 73 12 PRT Homo sapiens 73 Cys Gly Thr Arg Asp Trp Ile Glu Gly GluThr Leu 1 5 10 74 12 PRT Homo sapiens 74 Gly Thr Arg Asp Trp Ile Glu GlyGlu Thr Gly Cys 1 5 10 75 12 PRT Homo sapiens 75 Cys His Pro His Leu ProArg Ala Leu Met Leu Leu 1 5 10 76 12 PRT Homo sapiens 76 Cys Gly Thr HisPro His Leu Pro Arg Ala Leu Met 1 5 10 77 13 PRT Homo sapiens 77 Thr HisPro His Leu Pro Arg Ala Leu Met Arg Ser Cys 1 5 10 78 14 PRT Homosapiens 78 Gly Pro His Leu Pro Arg Ala Leu Met Arg Ser Ser Ser Cys 1 510 79 13 PRT Homo sapiens 79 Ala Pro Glu Trp Pro Gly Ser Arg Asp Lys ArgThr Cys 1 5 10 80 17 PRT Homo sapiens 80 Ala Pro Glu Trp Pro Gly Ser ArgAsp Lys Arg Thr Leu Ala Gly Gly 1 5 10 15 Cys 81 17 PRT Homo sapiens 81Cys Gly Gly Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr 1 5 1015 Leu 82 13 PRT Homo sapiens 82 Cys Thr Arg Lys Asp Arg Ser Gly Pro TrpGlu Pro Ala 1 5 10 83 11 PRT Homo sapiens 83 Cys Gly Ala Glu Trp Glu GlnLys Asp Glu Leu 1 5 10 84 11 PRT Homo sapiens 84 Ala Glu Trp Glu Gln LysAsp Glu Phe Ile Cys 1 5 10 85 9 PRT Homo sapiens 85 Gly Glu Gln Lys AspGlu Phe Ile Cys 1 5 86 10 PRT Homo sapiens 86 Cys Ala Glu Gly Glu GlnLys Asp Glu Leu 1 5 10 87 6 PRT Homo sapiens 87 Leu Phe Ile Arg Lys Ser1 5 88 7 PRT Homo sapiens 88 Pro Ser Lys Gly Thr Val Asn 1 5 89 23 PRTHomo sapiens 89 Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His Ser ThrThr Gln 1 5 10 15 Pro Arg Lys Thr Lys Gly Ser 20 90 6 PRT Homo sapiens90 Ser Val Asn Pro Gly Lys 1 5 91 13 PRT Homo sapiens 91 Cys Pro Glu TrpPro Gly Cys Arg Asp Lys Arg Thr Gly 1 5 10 92 13 PRT Homo sapiens 92 ThrPro Glu Trp Pro Gly Cys Arg Asp Lys Arg Cys Gly 1 5 10 93 14 PRT Homosapiens 93 Asp Pro Glu Trp Pro Gly Ser Arg Asp Lys Lys Gly Ser Cys 1 510 94 13 PRT Homo sapiens 94 Asp Trp Pro Gly Ser Arg Asp Lys Arg Lys GlySer Cys 1 5 10 95 19 PRT Homo sapiens 95 Asp Ala Thr Pro Glu Trp Pro GlySer Arg Asp Lys Arg Thr Leu Lys 1 5 10 15 Gly Ser Cys 96 13 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 96Cys Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Cys 1 5 10 97 16 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 97Cys Phe Ile Asn Lys Gln Met Ala Asp Leu Glu Leu Cys Pro Arg Glu 1 5 1015 98 16 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 98 Cys Phe Met Asn Lys Gln Leu Ala Asp Leu Glu Leu Cys Pro ArgGlu 1 5 10 15 99 22 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 99 Cys Leu Glu Asp Gly Gln Val Met Asp Val Asp LeuCys Pro Arg Glu 1 5 10 15 Ala Ala Glu Gly Asp Lys 20 100 20 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 100Cys Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Cys Gly Gly Ser 1 5 1015 Ser Gly Gly Pro 20 101 21 PRT Artificial Sequence Artificial variantof Homo sapiens IgE peptide 101 Cys Leu Glu Asp Gly Gln Val Met Asp ValAsp Cys Pro Arg Glu Ala 1 5 10 15 Ala Glu Gly Asp Lys 20 102 17 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 102Lys Cys Arg Glu Val Trp Leu Gly Glu Ser Glu Thr Ile Met Asp Cys 1 5 1015 Glu 103 17 PRT Artificial Sequence Artificial variant of Homo sapiensIgE peptide 103 Ala Cys Arg Glu Val Trp Leu Gly Glu Ser Glu Thr Ile MetAsp Cys 1 5 10 15 Asp 104 17 PRT Artificial Sequence Artificial variantof Homo sapiens IgE peptide 104 Ser Cys Arg Glu Val Trp Leu Gly Glu SerGlu Thr Val Met Asp Cys 1 5 10 15 Gly 105 17 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 105 Asn Cys Gln Asp LeuMet Leu Arg Glu Asp Ala Gly Cys Trp Ser Lys 1 5 10 15 Met 106 17 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 106Asp Cys Glu Glu Pro Met Cys Ser Pro Val Leu Leu Gln Gln Leu Lys 1 5 1015 Leu 107 13 PRT Artificial Sequence Artificial variant of Homo sapiensIgE peptide 107 Cys Phe Ile Asn Lys Gln Met Ala Asp Leu Glu Leu Cys 1 510 108 13 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 108 Cys Phe Met Asn Lys Gln Leu Ala Asp Leu Glu Leu Cys 1 5 10109 16 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 109 Lys Cys Arg Glu Val Trp Leu Gly Glu Ser Glu Thr Ile Met AspCys 1 5 10 15 110 17 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 110 His Cys Gln Gln Val Phe Phe Pro Gln Asp Tyr LeuTrp Cys Gln Arg 1 5 10 15 Gly 111 17 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 111 Ser Cys Arg Glu Val Trp Leu GlyGly Ser Glu Met Ile Met Asp Cys 1 5 10 15 Glu 112 17 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 112 Glu Cys AsnGln Asn Leu Ser Gly Ser Leu Arg His Val Asp Leu Asn 1 5 10 15 Cys 113 17PRT Artificial Sequence Artificial variant of Homo sapiens IgE peptide113 Asp Cys Glu Glu Pro Met Cys Ser Pro Val Leu Leu Gln Lys Leu Lys 1 510 15 Pro 114 17 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 114 Ser Cys Arg Glu Val Trp Leu Gly Gly Ser Glu MetIle Met Asp Cys 1 5 10 15 Glu 115 17 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 115 Arg Cys Asp Gln Gln Leu Pro ArgAsp Ser Tyr Thr Phe Cys Met Met 1 5 10 15 Ser 116 17 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 116 Ser Cys ProAla Phe Pro Arg Glu Gly Asp Leu Cys Ala Pro Pro Thr 1 5 10 15 Val 117 17PRT Artificial Sequence Artificial variant of Homo sapiens IgE peptide117 Phe Cys Pro Glu Pro Ile Cys Ser Pro Pro Leu Ser Arg Met Thr Leu 1 510 15 Ser 118 12 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 118 Val Cys Asp Glu Cys Val Ser Arg Glu Leu Ala Leu1 5 10 119 12 PRT Artificial Sequence Artificial variant of Homo sapiensIgE peptide 119 Trp Cys Leu Glu Pro Glu Cys Ala Pro Gly Leu Leu 1 5 10120 12 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 120 Val Cys Asp Glu Cys Val Ser Arg Glu Leu Ala Leu 1 5 10 12112 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 121 Asp Cys Leu Ser Lys Gly Gln Met Ala Asp Leu Cys 1 5 10 12212 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 122 Ser Cys Gln Gly Arg Glu Val Arg Arg Glu Cys Trp 1 5 10 12317 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 123 Trp Cys Arg Glu Val Trp Leu Gly Glu Ser Glu Thr Ile Met AspCys 1 5 10 15 Glu 124 17 PRT Artificial Sequence Artificial variant ofHomo sapiens IgE peptide 124 Ala Cys Arg Glu Val Trp Leu Gly Glu Ser GluThr Ile Met Asp Cys 1 5 10 15 Asp 125 17 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 125 Gly Cys Ala Glu ProLys Cys Trp Gln Ala Leu His Gln Lys Leu Lys 1 5 10 15 Pro 126 17 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 126Glu Cys Arg Gly Pro Asn Met Gln Met Gln Asp His Cys Pro Thr Thr 1 5 1015 Asp 127 17 PRT Artificial Sequence Artificial variant of Homo sapiensIgE peptide 127 Gln Cys Asn Ala Val Leu Glu Gly Leu Gln Met Val Asp HisCys Trp 1 5 10 15 Asn 128 17 PRT Artificial Sequence Artificial variantof Homo sapiens IgE peptide 128 His Cys Lys Asn Glu Phe Lys Lys Gly GlnTrp Thr Tyr Ser Cys Ser 1 5 10 15 Asp 129 17 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 129 Gln Cys Arg Gln PheVal Met Asn Gln Ser Glu Lys Glu Phe Gly Gln 1 5 10 15 Cys 130 17 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 130Asn Cys Phe Met Asn Lys Gln Leu Ala Asp Leu Glu Leu Cys Pro Arg 1 5 1015 Glu 131 17 PRT Artificial Sequence Artificial variant of Homo sapiensIgE peptide 131 Ser Cys Ala Tyr Thr Ala Gln Arg Gln Cys Ser Asp Val ProAsn Pro 1 5 10 15 Gly 132 19 PRT Artificial Sequence Artificial variantof Homo sapiens IgE peptide 132 Gly Cys Phe Met Asn Lys Gln Met Ala AspLeu Glu Leu Cys Pro Arg 1 5 10 15 Thr Ala Ala 133 19 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 133 Ala Cys PheMet Asn Lys Gln Met Ala Asp Leu Glu Leu Cys Pro Arg 1 5 10 15 Val AlaAla 134 19 PRT Artificial Sequence Artificial variant of Homo sapiensIgE peptide 134 Gly Cys Phe Ile Asn Lys Gln Leu Ala Asp Leu Glu Leu CysPro Arg 1 5 10 15 Val Ala Ala 135 19 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 135 Gly Cys Phe Met Asn Lys Gln LeuAla Asp Trp Glu Leu Cys Pro Arg 1 5 10 15 Ala Ala Ala 136 19 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 136Glu Cys Phe Met Asn Lys Gln Leu Ala Asp Ser Glu Leu Cys Pro Arg 1 5 1015 Val Ala Ala 137 19 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 137 Gly Cys Phe Met Asn Lys Gln Leu Ala Asp Pro GluLeu Cys Pro Arg 1 5 10 15 Glu Ala Glu 138 19 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 138 Gly Cys Phe Met AsnLys Gln Leu Val Asp Leu Glu Leu Cys Pro Arg 1 5 10 15 Gly Ala Ala 139 19PRT Artificial Sequence Artificial variant of Homo sapiens IgE peptide139 Gly Cys Phe Met Asn Lys Gln Leu Ala Asp Leu Glu Leu Cys Pro Arg 1 510 15 Glu Ala Ala 140 19 PRT Artificial Sequence Artificial variant ofHomo sapiens IgE peptide 140 Gly Cys Phe Met Asn Lys Gln Gln Ala Asp LeuGlu Leu Cys Pro Arg 1 5 10 15 Gly Ala Ala 141 19 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 141 Gly Cys Phe Ile AsnLys Gln Met Ala Asp Leu Glu Leu Cys Pro Arg 1 5 10 15 Glu Ala Ala 142 20PRT Artificial Sequence Artificial variant of Homo sapiens IgE peptide142 Cys Leu Glu Asp Gly Gln Val Met Asp Val Asp Cys Pro Arg Glu Ala 1 510 15 Ala Glu Gly Asp 20 143 21 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 143 Cys Leu Glu Asp Gly Gln Val MetAsp Val Asp Leu Cys Pro Arg Glu 1 5 10 15 Ala Ala Glu Gly Asp 20 144 17PRT Artificial Sequence Artificial variant of Homo sapiens IgE peptide144 Gln Cys Asn Ala Val Leu Glu Gly Leu Gln Met Val Asp His Cys Trp 1 510 15 Asn 145 17 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 145 Glu Cys Leu Lys Ile Glu Gln Gln Cys Ala Asp IleVal Glu Ile Pro 1 5 10 15 Arg 146 17 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 146 Ser Cys Ala Tyr Thr Ala Gln ArgGln Cys Ser Asp Val Pro Asn Pro 1 5 10 15 Gly 147 17 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 147 Glu Cys ArgGly Pro Asn Met Gln Met Gln Asp His Cys Pro Thr Thr 1 5 10 15 Asp 148 17PRT Artificial Sequence Artificial variant of Homo sapiens IgE peptide148 Glu Cys Leu Val Tyr Gly Gln Met Ala Asp Cys Ala Ala Gly Gly Trp 1 510 15 Pro 149 17 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 149 Gln Cys Arg Gln Phe Val Met Asn Gln Ser Glu LysGlu Phe Gly Gln 1 5 10 15 Cys 150 17 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 150 His Cys Lys Asn Glu Phe Lys LysGly Gln Trp Thr Tyr Ser Cys Ser 1 5 10 15 Asp 151 12 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 151 Cys Ala ProGly Met Gly Cys Trp Glu Ser Val Lys 1 5 10 152 17 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 152 Ser Cys ArgGlu Val Trp Leu Gly Gly Ser Glu Met Ile Met Asp Cys 1 5 10 15 Glu 153 17PRT Artificial Sequence Artificial variant of Homo sapiens IgE peptide153 Ser Cys Pro Ala Phe Pro Arg Glu Gly Asp Leu Cys Ala Pro Pro Thr 1 510 15 Val 154 17 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 154 Phe Cys Pro Glu Pro Ile Cys Ser Pro Pro Leu SerArg Met Thr Leu 1 5 10 15 Ser 155 17 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 155 Glu Cys Asn Gln Asn Leu Ser GlySer Leu Arg His Val Asp Leu Asn 1 5 10 15 Cys 156 17 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 156 Arg Cys AspGln Gln Leu Pro Arg Asp Ser Tyr Thr Phe Cys Met Met 1 5 10 15 Ser 157 17PRT Artificial Sequence Artificial variant of Homo sapiens IgE peptide157 His Cys Gln Gln Val Phe Phe Pro Gln Asp Tyr Leu Trp Cys Gln Arg 1 510 15 Gly 158 17 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 158 Asp Cys Glu Glu Pro Met Cys Ser Pro Val Leu LeuGln Lys Leu Lys 1 5 10 15 Pro 159 17 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 159 Asn Cys Gln Asp Gln Met Leu ArgGlu Asp Ala Gly Cys Trp Ser Lys 1 5 10 15 Ile 160 17 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 160 His Cys GluGlu Pro Glu Tyr Ser Pro Ala Thr Arg Val Phe Cys Gly 1 5 10 15 Arg 161 17PRT Artificial Sequence Artificial variant of Homo sapiens IgE peptide161 Ala Cys Phe Ser Arg Asn Gly Gln Val Thr Asp Val Pro His Ser Cys 1 510 15 Tyr 162 17 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 162 Lys Cys Pro Thr Tyr Pro Lys Pro Asn Asp Arg CysLeu Trp Pro Val 1 5 10 15 Pro 163 17 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 163 Tyr Cys Pro Lys Tyr Pro Leu GluGly Asp Cys Leu Leu Asp Asn Asp 1 5 10 15 Tyr 164 17 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 164 Arg Cys GluGlu Trp Leu Cys Ile Pro Pro Ala Pro Ala Phe Ala Pro 1 5 10 15 Pro 165 17PRT Artificial Sequence Artificial variant of Homo sapiens IgE peptide165 Thr Cys Gly Gln Ser Glu Leu Arg Cys Ala Ser Leu Glu Thr His His 1 510 15 Val 166 16 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 166 Asn Cys Asn Asp Asn Pro Met Leu Asp Cys Met ProAla Trp Ser Ser 1 5 10 15 167 12 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 167 Ser Cys Gln Gly Arg Glu Val ArgArg Glu Cys Trp 1 5 10 168 12 PRT Artificial Sequence Artificial variantof Homo sapiens IgE peptide 168 Val Cys Asp Glu Cys Val Ser Arg Glu LeuAla Leu 1 5 10 169 12 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 169 Trp Cys Leu Glu Pro Glu Cys Ala Pro Gly Leu Leu1 5 10 170 12 PRT Artificial Sequence Artificial variant of Homo sapiensIgE peptide 170 Asp Cys Leu Ser Lys Gly Gln Met Ala Asp Leu Cys 1 5 10171 12 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 171 Val Cys Asp Glu Cys Val Ser Arg Glu Leu Ala Leu 1 5 10 17212 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 172 Gly Cys Pro Thr Trp Pro Arg Val Gly Asp His Cys 1 5 10 17312 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 173 Arg Cys Gln Ser Ala Arg Val Val Pro Glu Cys Trp 1 5 10 17412 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 174 Ser Cys Ala Pro Ser Gly Asp Cys Gly Tyr Lys Gly 1 5 10 17512 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 175 Gly Cys Pro Met Trp Pro Gln Pro Asp Asp Glu Cys 1 5 10 17612 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 176 Glu Cys Pro Arg Trp Pro Leu Met Gly Asp Gly Cys 1 5 10 17712 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 177 Gly Cys Gln Val Gly Glu Leu Val Trp Cys Arg Glu 1 5 10 17812 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 178 Gln Cys Val Arg Asp Gly Thr Arg Lys Val Cys Met 1 5 10 17912 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 179 Thr Cys Leu Val Asp Arg Gln Glu Ser Asp Val Cys 1 5 10 18012 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 180 Asp Cys Val Val Asp Gly Asp Arg Leu Val Cys Leu 1 5 10 18112 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 181 Arg Cys Glu Gln Gly Ala Leu Arg Cys Val Gly Glu 1 5 10 18212 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 182 Val Cys Pro Pro Gly Trp Lys Asn Leu Gly Cys Asn 1 5 10 18312 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 183 Met Cys Gln Gly Trp Glu Ile Val Ser Glu Cys Trp 1 5 10 18425 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 184 Ala Asp Gly Ala Gly Cys Phe Met Asn Lys Gln Met Ala Asp LeuGlu 1 5 10 15 Leu Cys Pro Arg Glu Ala Ala Glu Ala 20 25 185 25 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 185Ala Asp Gly Ala Gly Cys Phe Met Asn Lys Gln Met Ala Asp Leu Glu 1 5 1015 Leu Cys Pro Arg Thr Ala Ala Glu Ala 20 25 186 25 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 186 Ala Asp GlyAla Ala Cys Phe Met Asn Lys Gln Met Ala Asp Leu Glu 1 5 10 15 Leu CysPro Arg Val Ala Ala Glu Ala 20 25 187 25 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 187 Ala Asp Gly Ala GlyCys Phe Ile Asn Lys Gln Leu Ala Asp Leu Glu 1 5 10 15 Leu Cys Pro ArgVal Ala Ala Glu Ala 20 25 188 25 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 188 Ala Asp Gly Ala Gly Cys Phe IleAsn Lys Gln Leu Ala Asp Leu Glu 1 5 10 15 Leu Cys Pro Arg Glu Ala AlaGlu Ala 20 25 189 25 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 189 Ala Asp Gly Ala Gly Cys Phe Met Asn Lys Gln LeuAla Asp Leu Glu 1 5 10 15 Met Cys Pro Arg Asp Asp Ala Glu Ala 20 25 19025 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 190 Ala Asp Gly Ala Gly Cys Phe Met Asn Lys Gln Leu Ala Asp ProGlu 1 5 10 15 Leu Cys Pro Arg Glu Ala Glu Glu Ala 20 25 191 25 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 191Ala Asp Gly Ala Gly Cys Phe Met Asn Lys Gln Leu Val Asp Leu Glu 1 5 1015 Leu Cys Pro Arg Gly Ala Ala Glu Ala 20 25 192 25 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 192 Ala Asp GlyAla Gly Cys Phe Met Asn Asn Gln Leu Ala Asp Trp Glu 1 5 10 15 Leu CysPro Arg Ala Ala Ala Glu Ala 20 25 193 25 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 193 Ala Asp Gly Ala GlyCys Phe Met Asn Lys Gln Met Ala Asp Trp Glu 1 5 10 15 Met Cys Pro ArgAla Ala Ala Glu Ala 20 25 194 25 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 194 Ala Asp Gly Ala Gly Cys Phe MetAsn Lys Gln Gln Ala Asp Leu Glu 1 5 10 15 Leu Cys Pro Arg Gly Ala AlaGlu Ala 20 25 195 25 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 195 Ala Asp Gly Ala Glu Cys Phe Met Asn Lys Gln LeuAla Asp Ser Glu 1 5 10 15 Leu Cys Pro Arg Val Ala Ala Glu Ala 20 25 19625 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 196 Ala Asp Gly Ala Gly Cys Phe Met Asn Lys Gln Leu Ala Asp LeuGlu 1 5 10 15 Leu Cys Pro Arg Glu Ala Ala Glu Ala 20 25 197 25 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 197Ala Asp Gly Ala Gly Cys Phe Ile Asn Met Gln Met Ala Asp Gln Glu 1 5 1015 Leu Cys Pro Arg Ala Ala Ala Glu Ala 20 25 198 25 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 198 Ala Asp GlyAla Gly Cys Phe Ile Asn Lys Gln Met Ser Asp Phe Glu 1 5 10 15 Leu CysPro Arg Glu Ala Gly Glu Ala 20 25 199 25 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 199 Ala Asp Gly Ala GlyCys Phe Ile Asn Lys Gln Met Ala Asp Leu Glu 1 5 10 15 Leu Cys Thr ArgGlu Ala Ala Glu Ala 20 25 200 25 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 200 Ala Asp Gly Ala Gly Cys Phe IleAsn Lys Gln Met Ala Asp Leu Glu 1 5 10 15 Leu Cys Pro Arg Gln Ala AlaGlu Ala 20 25 201 25 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 201 Ala Asp Gly Ala Gly Cys Phe Ile Asn Asn Gln MetAla Asp Leu Glu 1 5 10 15 Leu Cys Pro Arg Gly Gly Ala Glu Ala 20 25 20225 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 202 Ala Asp Gly Ala Gly Cys Phe Ile Asn Lys Gln Met Ala Asp TrpGlu 1 5 10 15 Leu Cys Pro Arg Glu Gly Ala Glu Ala 20 25 203 25 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 203Ala Asp Gly Ala Gly Cys Phe Ile Asn Lys Gln Met Ala Asp Leu Glu 1 5 1015 Leu Cys Pro Ser Gln Ala Ala Glu Ala 20 25 204 25 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 204 Ala Asp GlyAla Gly Cys Phe Ile Asn Lys Gln Met Ala Asp Leu Glu 1 5 10 15 Leu CysPro Arg Glu Gly Ala Glu Ala 20 25 205 25 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 205 Ala Asp Gly Ala GlyCys Phe Ile Asn Lys Gln Met Ala Asp Ser Glu 1 5 10 15 Leu Cys Pro ArgGlu Pro Ala Glu Ala 20 25 206 25 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 206 Ala Asp Gly Ala Gly Cys Phe IleLys Lys Gln Met Ala Asp Leu Glu 1 5 10 15 Leu Cys Pro Arg Glu Ala TrpGlu Ala 20 25 207 25 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 207 Ala Asp Gly Ala Glu Cys Phe Ile Asn Lys Gln MetAla Asp Arg Glu 1 5 10 15 Leu Cys Ala Arg Glu Val Ala Glu Ala 20 25 20825 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 208 Ala Asp Gly Ala Gly Cys Phe Ile Asp Lys Gln Met Ala Asp LeuGlu 1 5 10 15 Leu Cys Pro Arg Ala Ala Ala Glu Ala 20 25 209 25 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 209Ala Asp Gly Ala Gly Cys Phe Ile Asn Lys Gln Met Ala Asp Leu Glu 1 5 1015 Leu Cys Arg Arg Glu Ala Gly Glu Ala 20 25 210 25 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 210 Ala Asp GlyAla Gly Cys Phe Lys Asn Lys Gln Met Val Asp Ser Glu 1 5 10 15 Leu CysAla Arg Gln Ala Ala Glu Ala 20 25 211 25 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 211 Ala Asp Gly Ala GlyCys Phe Gln Asn Lys Gln Met Ala Asp Leu Glu 1 5 10 15 Leu Cys Pro ArgGlu Ala Ala Glu Ala 20 25 212 25 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 212 Ala Asp Gly Ala Glu Cys Phe IleAsn Lys Gln Arg Ala Asp Leu Glu 1 5 10 15 Leu Cys Pro Gly Glu Ala AlaGlu Ala 20 25 213 25 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 213 Ala Asp Gly Ala Gly Cys Phe Ile Asn Lys Gln MetAla Asp Ser Glu 1 5 10 15 Leu Cys Pro Ala Ala Ala Ala Glu Ala 20 25 21425 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 214 Ala Asp Gly Ala Gly Cys Phe Ile Asn Arg Gln Met Ala Asp ProGlu 1 5 10 15 Leu Cys Pro Arg Glu Ala Ala Glu Ala 20 25 215 25 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 215Ala Asp Gly Ala Gly Cys Phe Ile Glu Lys Gln Met Ala Asp Met Glu 1 5 1015 Leu Cys Gln Ala Arg Ala Ala Glu Ala 20 25 216 25 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 216 Ala Asp GlyAla Gly Cys Phe Ile Asn Lys Gln Met Ala Asp Trp Glu 1 5 10 15 Leu CysPro Arg Glu Ala Ala Glu Ala 20 25 217 25 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 217 Ala Asp Gly Ala GlyCys Phe Ile Asn Asn Gln Met Ala Asp Leu Glu 1 5 10 15 Leu Cys Pro ArgGlu Ala Ala Glu Ala 20 25 218 25 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 218 Ala Asp Gly Ala Gly Cys Phe IleGlu Lys Gln Met Ala Asp Met Glu 1 5 10 15 Leu Cys Gln Arg Glu Thr AlaGlu Ala 20 25 219 25 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 219 Ala Asp Gly Ala Gly Cys Phe Ile Asn Lys Gln MetAla Asp Met Glu 1 5 10 15 Leu Cys Pro Arg Glu Ala Ala Glu Ala 20 25 22025 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 220 Ala Asp Gly Ala Gly Cys Phe Ile Asn Lys Gln Met Ala Asp LeuGlu 1 5 10 15 Leu Cys Pro Arg Glu Ala Ala Glu Ala 20 25 221 25 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 221Ala Asp Gly Ala Gly Cys Phe Arg Asn Lys Gln Met Ala Asp Leu Glu 1 5 1015 Leu Cys Pro Arg Glu Ala Ala Glu Ala 20 25 222 25 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 222 Ala Asp GlyAla Gly Cys Phe Ile Asn Lys Gln Met Ala Asp Leu Glu 1 5 10 15 Leu CysPro Ala Arg Ala Ala Glu Ala 20 25 223 25 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 223 Ala Asp Gly Ala GlyCys Phe Ile Asn Arg Gln Leu Ala Asp Met Glu 1 5 10 15 Leu Cys Ser ArgGly Ala Ala Glu Ala 20 25 224 25 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 224 Ala Asp Gly Ala Glu Cys Phe IleAsn Arg Gln Met Ala Asp Leu Glu 1 5 10 15 Leu Cys Gly Arg Glu Ala AlaGlu Ala 20 25 225 25 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 225 Ala Asp Gly Ala Gly Cys Phe Ile Ser Pro Gln LeuAla Asp Trp Lys 1 5 10 15 Arg Cys Met Arg Glu Ala Ala Glu Ala 20 25 22625 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 226 Ala Asp Gly Ala Gly Cys Ser Ile His Thr Gln Met Ala Asp TrpGlu 1 5 10 15 Arg Cys Leu Arg Glu Gly Ala Glu Ala 20 25 227 25 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 227Ala Asp Gly Ala Gly Cys Ser Ile His Arg Gln Met Ala Asp Trp Glu 1 5 1015 Arg Cys Leu Arg Glu Gly Ala Glu Ala 20 25 228 16 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 228 Cys Ser SerCys Asp Gly Gly Gly His Lys Pro Pro Thr Ile Gln Cys 1 5 10 15 229 20 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 229Cys Leu Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile 1 5 1015 Gln Leu Leu Cys 20 230 15 PRT Artificial Sequence Artificial variantof Homo sapiens IgE peptide 230 Ala Pro Cys Trp Pro Gly Ser Arg Asp CysArg Thr Leu Ala Gly 1 5 10 15 231 16 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 231 Ala Cys Pro Glu Trp Pro Gly SerArg Asp Arg Cys Thr Leu Ala Gly 1 5 10 15 232 17 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 232 Cys Ala Thr Pro GluTrp Pro Gly Ser Arg Asp Lys Arg Thr Leu Cys 1 5 10 15 Gly 233 16 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 233Cys Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Cys Gly 1 5 1015 234 13 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 234 Thr Pro Cys Trp Pro Gly Ser Arg Asp Lys Arg Cys Gly 1 5 10235 19 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 235 Cys Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser ProThr 1 5 10 15 Ile Thr Cys 236 18 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 236 Cys Ser Arg Pro Ser Pro Phe AspLeu Phe Ile Arg Lys Ser Pro Thr 1 5 10 15 Ile Cys 237 17 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 237 Cys Ser ArgPro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr 1 5 10 15 Cys 238 16PRT Artificial Sequence Artificial variant of Homo sapiens IgE peptide238 Cys Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Cys 1 510 15 239 15 PRT Artificial Sequence Artificial variant of Homo sapiensIgE peptide 239 Cys Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser ProCys 1 5 10 15 240 16 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 240 Cys Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg LysSer Pro Thr Cys 1 5 10 15 241 17 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 241 Cys Arg Pro Ser Pro Phe Asp LeuPhe Ile Arg Lys Ser Pro Thr Ile 1 5 10 15 Cys 242 18 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 242 Cys Arg ProSer Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile 1 5 10 15 Thr Cys243 17 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 243 Cys Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr IleThr 1 5 10 15 Cys 244 16 PRT Artificial Sequence Artificial variant ofHomo sapiens IgE peptide 244 Cys Pro Ser Pro Phe Asp Leu Phe Ile Arg LysSer Pro Thr Ile Cys 1 5 10 15 245 15 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 245 Cys Pro Ser Pro Phe Asp Leu PheIle Arg Lys Ser Pro Thr Cys 1 5 10 15 246 14 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 246 Cys Pro Ser Pro PheAsp Leu Phe Ile Arg Lys Ser Pro Cys 1 5 10 247 20 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 247 Cys Tyr AlaPhe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg 1 5 10 15 Thr LeuAla Cys 20 248 19 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 248 Cys Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly SerArg Asp Lys Arg 1 5 10 15 Thr Leu Cys 249 18 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 249 Cys Tyr Ala Phe AlaThr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg 1 5 10 15 Thr Cys 250 17 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 250Cys Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg 1 5 1015 Cys 251 16 PRT Artificial Sequence Artificial variant of Homo sapiensIgE peptide 251 Cys Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp LysArg Cys 1 5 10 15 252 17 PRT Artificial Sequence Artificial variant ofHomo sapiens IgE peptide 252 Cys Ala Phe Ala Thr Pro Glu Trp Pro Gly SerArg Asp Lys Arg Thr 1 5 10 15 Cys 253 18 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 253 Cys Ala Phe Ala ThrPro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr 1 5 10 15 Leu Cys 254 19 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 254Cys Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr 1 5 1015 Leu Ala Cys 255 18 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 255 Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg AspLys Arg Thr Leu 1 5 10 15 Ala Cys 256 17 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 256 Cys Phe Ala Thr ProGlu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu 1 5 10 15 Cys 257 16 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 257Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Cys 1 5 1015 258 15 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 258 Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Cys1 5 10 15 259 17 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 259 Cys Thr Trp Ser Arg Ala Ser Gly Lys Pro Val AsnHis Ser Thr Arg 1 5 10 15 Cys 260 16 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 260 Cys Thr Trp Ser Arg Ala Ser GlyLys Pro Val Asn His Ser Thr Cys 1 5 10 15 261 15 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 261 Cys Thr Trp Ser ArgAla Ser Gly Lys Pro Val Asn His Ser Cys 1 5 10 15 262 14 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 262 Cys Thr TrpSer Arg Ala Ser Gly Lys Pro Val Asn His Cys 1 5 10 263 13 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 263 Cys Trp SerArg Ala Ser Gly Lys Pro Val Asn His Cys 1 5 10 264 14 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 264 Cys Trp SerArg Ala Ser Gly Lys Pro Val Asn His Ser Cys 1 5 10 265 15 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 265 Cys Trp SerArg Ala Ser Gly Lys Pro Val Asn His Ser Thr Cys 1 5 10 15 266 16 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 266Cys Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Cys 1 5 1015 267 15 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 267 Cys Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Cys1 5 10 15 268 14 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 268 Cys Ser Arg Ala Ser Gly Lys Pro Val Asn His SerThr Cys 1 5 10 269 13 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 269 Cys Ser Arg Ala Ser Gly Lys Pro Val Asn His SerCys 1 5 10 270 12 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 270 Cys Ser Arg Ala Ser Gly Lys Pro Val Asn His Cys1 5 10 271 17 PRT Artificial Sequence Artificial variant of Homo sapiensIgE peptide 271 Cys Gln Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala ArgHis Ser 1 5 10 15 Cys 272 16 PRT Artificial Sequence Artificial variantof Homo sapiens IgE peptide 272 Cys Gln Trp Leu His Asn Glu Val Gln LeuPro Asp Ala Arg His Cys 1 5 10 15 273 15 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 273 Cys Gln Trp Leu HisAsn Glu Val Gln Leu Pro Asp Ala Arg Cys 1 5 10 15 274 14 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 274 Cys Gln TrpLeu His Asn Glu Val Gln Leu Pro Asp Ala Cys 1 5 10 275 13 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 275 Cys Trp LeuHis Asn Glu Val Gln Leu Pro Asp Ala Cys 1 5 10 276 14 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 276 Cys Trp LeuHis Asn Glu Val Gln Leu Pro Asp Ala Arg Cys 1 5 10 277 15 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 277 Cys Trp LeuHis Asn Glu Val Gln Leu Pro Asp Ala Arg His Cys 1 5 10 15 278 16 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 278Cys Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His Ser Cys 1 5 1015 279 15 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 279 Cys Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His Ser Cys1 5 10 15 280 14 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 280 Cys Leu His Asn Glu Val Gln Leu Pro Asp Ala ArgHis Cys 1 5 10 281 13 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 281 Cys Leu His Asn Glu Val Gln Leu Pro Asp Ala ArgCys 1 5 10 282 12 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 282 Cys Leu His Asn Glu Val Gln Leu Pro Asp Ala Cys1 5 10 283 17 PRT Artificial Sequence Artificial variant of Homo sapiensIgE peptide 283 Cys Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro CysGly Ser 1 5 10 15 Lys 284 18 PRT Artificial Sequence Artificial variantof Homo sapiens IgE peptide 284 Cys Pro Ser Pro Phe Asp Leu Phe Ile ArgLys Ser Pro Thr Cys Gly 1 5 10 15 Ser Lys 285 20 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 285 Phe Ala Gly Cys SerArg Ala Ser Gly Lys Pro Val Asn His Cys Gly 1 5 10 15 Ala Ala Glu Gly 20286 21 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 286 Phe Ala Gly Cys Ser Arg Ala Ser Gly Lys Pro Val Asn His SerCys 1 5 10 15 Gly Ala Ala Glu Gly 20 287 22 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 287 Phe Ala Gly Cys SerArg Ala Ser Gly Lys Pro Val Asn His Ser Thr 1 5 10 15 Cys Gly Ala AlaGlu Gly 20 288 23 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 288 Phe Ala Gly Cys Ser Arg Ala Ser Gly Lys Pro ValAsn His Ser Thr 1 5 10 15 Arg Cys Gly Ala Ala Glu Gly 20 289 15 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 289Cys Ser Arg Ala Ser Gly Lys Pro Val Asn His Cys Gly Ser Lys 1 5 10 15290 16 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 290 Cys Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Cys Gly SerLys 1 5 10 15 291 17 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 291 Cys Ser Arg Ala Ser Gly Lys Pro Val Asn His SerThr Cys Gly Ser 1 5 10 15 Lys 292 23 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 292 Phe Ala Gly Cys Phe Ala Thr ProGlu Trp Pro Gly Ser Arg Asp Lys 1 5 10 15 Arg Cys Gly Ala Ala Glu Gly 20293 24 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 293 Phe Ala Gly Cys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg AspLys 1 5 10 15 Arg Thr Cys Gly Ala Ala Glu Gly 20 294 25 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 294 Phe Ala GlyCys Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys 1 5 10 15 Arg ThrLeu Cys Gly Ala Ala Glu Gly 20 25 295 26 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 295 Phe Ala Gly Cys PheAla Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys 1 5 10 15 Arg Thr Leu AlaCys Gly Ala Ala Glu Gly 20 25 296 15 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 296 Cys Pro Glu Trp Pro Gly Ser ArgAsp Lys Arg Cys Gly Ser Lys 1 5 10 15 297 13 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 297 Cys Trp Pro Gly SerArg Asp Lys Arg Cys Gly Ser Lys 1 5 10 298 17 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 298 Cys Pro Glu Trp ProGly Ser Arg Asp Lys Arg Cys Gly Ala Ala Glu 1 5 10 15 Gly 299 20 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 299Phe Ala Gly Cys Leu His Asn Glu Val Gln Leu Pro Asp Ala Cys Gly 1 5 1015 Ala Ala Glu Gly 20 300 21 PRT Artificial Sequence Artificial variantof Homo sapiens IgE peptide 300 Phe Ala Gly Cys Leu His Asn Glu Val GlnLeu Pro Asp Ala Arg Cys 1 5 10 15 Gly Ala Ala Glu Gly 20 301 22 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 301Phe Ala Gly Cys Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His 1 5 1015 Cys Gly Ala Ala Glu Gly 20 302 23 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 302 Phe Ala Gly Cys Leu His Asn GluVal Gln Leu Pro Asp Ala Arg His 1 5 10 15 Ser Cys Gly Ala Ala Glu Gly 20303 20 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 303 Phe Ala Gly Cys Leu His Asn Glu Val Gln Leu Pro Asp Ala SerGly 1 5 10 15 Ala Ala Glu Gly 20 304 14 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 304 Cys Pro Glu Trp ProGly Ser Arg Asp Arg Cys Gly Ser Lys 1 5 10 305 13 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 305 Cys Trp ProGly Ser Arg Asp Arg Arg Cys Gly Ser Lys 1 5 10 306 20 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 306 Cys Asp SerAsn Pro Arg Gly Val Ser Ala Ala Asp Ser Asn Pro Arg 1 5 10 15 Gly ValSer Cys 20 307 15 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 307 Cys Leu Val Val Asp Leu Ala Pro Ser Lys Gly ThrVal Asn Cys 1 5 10 15 308 9 PRT Artificial Sequence Artificial variantof Homo sapiens IgE peptide 308 Cys Lys Gln Arg Asn Gly Thr Leu Cys 1 5309 13 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 309 Cys Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr Val Cys 1 5 10310 8 PRT Artificial Sequence Artificial variant of Homo sapiens IgEpeptide 310 Cys His Pro His Leu Pro Arg Cys 1 5 311 10 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 311 Cys Thr HisPro His Leu Pro Arg Ala Cys 1 5 10 312 12 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 312 Cys Val Thr His ProHis Leu Pro Arg Ala Leu Cys 1 5 10 313 14 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 313 Cys Arg Val Thr HisPro His Leu Pro Arg Ala Leu Met Cys 1 5 10 314 16 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 314 Cys Xaa ArgVal Thr His Pro His Leu Pro Arg Ala Leu Met Arg Cys 1 5 10 15 315 18 PRTArtificial Sequence Artificial variant of Homo sapiens IgE peptide 315Cys Gln Xaa Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met Arg 1 5 1015 Ser Cys 316 20 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 316 Cys Tyr Gln Xaa Arg Val Thr His Pro His Leu ProArg Ala Leu Met 1 5 10 15 Arg Ser Thr Cys 20 317 12 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 317 Cys Pro GluTrp Pro Gly Ser Arg Asp Lys Arg Cys 1 5 10 318 9 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 318 Cys Arg Gln Arg AsnGly Thr Leu Cys 1 5 319 13 PRT Artificial Sequence Artificial variant ofHomo sapiens IgE peptide 319 Cys Glu Glu Arg Gln Arg Asn Gly Thr Leu ThrVal Cys 1 5 10 320 16 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 320 Cys Met Arg Val Thr His Pro His Leu Pro Arg AlaLeu Met Arg Cys 1 5 10 15 321 18 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 321 Cys Gln Met Arg Val Thr His ProHis Leu Pro Arg Ala Leu Met Arg 1 5 10 15 Ser Cys 322 20 PRT ArtificialSequence Artificial variant of Homo sapiens IgE peptide 322 Cys Tyr GlnMet Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met 1 5 10 15 Arg SerThr Cys 20 323 16 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 323 Ala Cys Pro Glu Trp Pro Gly Ser Arg Asp Arg CysThr Leu Ala Gly 1 5 10 15 324 14 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 324 Gly Gly Cys Leu Glu Asp Gly GlnVal Met Asp Val Asp Cys 1 5 10 325 13 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 325 Cys Leu Glu Asp Gly Gln Val MetAsp Cys Gly Ser Lys 1 5 10 326 16 PRT Artificial Sequence Artificialvariant of Homo sapiens IgE peptide 326 Cys Leu Glu Asp Gly Gln Val MetAsp Val Asp Leu Cys Gly Ser Lys 1 5 10 15 327 22 PRT Artificial SequenceArtificial variant of Homo sapiens IgE peptide 327 Cys Leu Glu Asp GlyGln Val Met Asp Val Asp Leu Cys Pro Arg Glu 1 5 10 15 Ala Ala Glu GlyAsp Lys 20 328 20 PRT Artificial Sequence Artificial variant of Homosapiens IgE peptide 328 Cys Leu Glu Asp Gly Gln Val Met Asp Val Asp LeuCys Gly Gly Ser 1 5 10 15 Ser Gly Gly Lys 20

1. A process for the manufacture of a vaccine immunogen comprisingconjugating a disulphide bridge cyclised peptide to an immunogeniccarrier comprising, (a) adding to a disulphide cyclised peptide a moietycomprising a reactive group which is capable of forming thio-etherlinkages with thiol bearing carriers, and (b) reacting the activatedcyclised peptide thus formed with a thiol bearing immunogenic carrier.2. A process as claimed in claim 1 wherein the reactive group capable offorming thio-ether linkages with thiol bearing carriers is a maleimidegroup.
 3. A process as claimed in claim 1 wherein the disulphide bridgecyclised peptide is derived from human IgE.
 4. A process as claimed inclaim 3, wherein the human IgE peptide is selected from any one of SEQID NOs. 1 to
 328. 5. A process as claimed in claim 1, wherein thecarrier is selected from Haemophilus Influenzae Protein D, BSA, Keyholelimpet Haemocyanin (KLH), serum albumins such as bovine serum albumin(BSA), inactivated bacterial toxins such as tetanus or diptheria toxins(TT and DT), or recombinant fragments thereof (for example, Domain 1 ofFragment C of TT, or the translocation domain of DT), or the purifiedprotein derivative of tuberculin (PPD).
 6. A disulphide bridge cyclisedIgE peptide maleimide derivative.
 7. Use of a peptide derivative asclaimed in claim 6, in the manufacture of a medicament for the treatmentof allergy.
 8. A conjugate suitable for use in a vaccine, of formula(I):

wherein, carrier is an immunogenic carrier molecule, X is either alinker or a bond, Y is either a linker or a bond, and P is a disulphidebridge cyclised peptide.
 9. A conjugate as claimed in claim 8 wherein Pis selected from the following group SEQ ID NO.s 99, 304, 305, 306, 307,308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,322, 323, 324, 325, 326, 327, and
 328. 10. A vaccine compositioncomprising the product of the process claimed in any one of claims 1 to5, and a suitable adjuvant or carrier.
 11. A vaccine compositioncomprising a conjugate as claimed in claim 8 or 9, and a suitableadjuvant or carrier.
 12. A vaccine as claimed in claim 10 or 11, whereinthe vaccine is an allergy vaccine.
 13. A conjugate as claimed in claim 8for the treatment of allergy.